Brochure - Biological NMR - University of Southampton

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South West Structural Biology
Consortium Meeting 2012


16
th
-
17
th

July 2012

University of Southampton, UK

SWSBC 2012 University of Southampton


2

Table of Contents


Table of Contents

________________________________
_____________________

2

Campus Map

________________________________
________________________

3

Welcome to SWSBC 2012

________________________________
______________

4

Conferen
ce Information

________________________________
_______________

4

Conference Dinner

________________________________
____________________

4

Accommodation and Parking

________________________________
___________

4

SWSBC Programme

________________________________
___________________

6

Additional
Events on Tuesday 17th afternoon

______________________________

8

1) Southampton Diffraction Centre and Rigaku Europe Meeting

_________________________

8

2) NMR Discussion Group

________________________________
______________________

8

Sponsors

________________________________
____________________________

9

Talk Abstracts

________________________________
_______________________

11

Poster Abstracts

________________________________
_____________________

29

Delegate List

________________________________
________________________

49



SWSBC 2012 University of Southampton


3

Campus Map



SWSBC 2012 University of Southampton


4

Welcome to SWSBC 2012


Welcome to the 11
th

South West Structural Biology Consortium meeting at the
University of Southampton
. We hope you enjoy
the meeting and your stay in
Southampton.



Conference Information


The main meeting sessions will take place in
the
L
ecture
T
heatre
in Building 67
(Nig
h
t
ingale Building)
which is directly off the main foyer. Registration takes place in
the foyer where you will be issued with keys to your accommodation.

Please return
your keys to the
Glen Eyre
reception desk
on

Tuesday morning prior to the first
session starting at 9.00am. Luggage can be stored securely in
Building 67

(at the rear
of the lecture theatre or in Teaching Room 4)

where the conference is being held.


Posters and sponsors will be exhibiting in
B67
rooms 1003 and 1007
next to the
lecture theatre
.

T
ea

and
coffee
will be

served

here

during breaks
.


Breakfast
is

served in the
Terrace
restaurant

(B38). Lunches

and the conference
dinner will be s
erved in
locations
as indicated
on the map
overleaf
and listed in the
programme.



Conference Dinner


The conference dinner
on Monday
starts with

a wine reception

at 7.15pm

with live
Jazz music
.
The conference dinner will

take place in Garden Court
(B
38)
.



Accommodation and Parking


Accommodation
at

Glen Eyre Hall
is about 10

minutes walk north of the main
university campus. Delegates arrivi
ng on Sunday 15
th

July
must
collect their keys
directly from the Hall reception desk
(open

24h a day
)
.

Delegates arriving on Monday
16
th

July will have their keys allocated with their conference pack at registration in
The Nightingale

Building (B67).


Parking permits must be displayed in all vehicles
. Vehicles

should be left at Glen Eyre
H
all for the duration of the stay.







SWSBC 2012 University of Southampton


5

Accommodation map of Glen Eyre Hall for SWSBC delegates


Hartley Grove Blocks B and C are reserved

for SWSBC.

















SWSBC 2012 University of Southampton


6

SWSBC Programme

Monday July 16th 2012


10.00
-

11.0
0:

Registration, poster set up and sponsor exhibits in Nightingale Building
with tea & coffee.


11.0
0
-

12.40: Session 1
"Enzymes"

11.00
-

11.25 Jennifer Littlechild

(
University of Exeter)

Thermophilic Enzymes and Applications in Commercial Biocatalysis

11.25
-

11.50 Paul James

(University of Exeter)

Human Peroxiredoxin and Oxidative Stress

11.50
-

12.15 James Spencer

(University of Bristol)

The Basis for Carbapenem H
ydrolysis by Class A β
-
Lactamases: A Combined
Investigation using Crystallography and Simulations

12.15
-

12.40 Richard Birkinshaw

(University of Bristol)

The Moraxella catarrhalis DOXP Reductoisomerase


12.40
-

14.00: Lunch in new Life Science Building
Observatory

(B85, level 7)


14.
00
-

15.4
0: Session 2
"Molecular Microbiology"

14.00
-

14.25 Alice Robson

(University of Bristol)

Structural Characterisation of the Adhesion AspA, a Key Determinant of
Biofilm Formation in Streptococcus pyogenes

14.25
-

14.
50 Marisa Till

(University of Bristol)

Decoding the Biosynthesis of the Antibiotic Mupirocin
-

The Role of MupS

14.50
-

15.15 Sunny Singh

(Queen Mary London & University of Sussex)

LcrH, a Class II Chaperone from Type Three Secretion System, Has a Highl
y
Flexible Native Structure

15.15
-

15.40 Francis Atanu

(
University of Reading
)

Computational
and
E
xpression
S
tudies of the Chlamydia pneumoniae Major
Outer Membrane Protein (MOMP)


15.40
-

16.45
: Poster session with tea, coffee and sponsor exhibits


16.45

-

18.00: Session 3
"
New Technologies,
Extracellular Matrix and Fibres"

16.45
-

17.10 Marcus Winter

(Agilent Technologies)

Macromolecular Applications with the New, Brighter Nova X
-
ray Source from
Agilent Technologies

17.10
-

17.35 Garrick Taylor

(Uni
versity of Southampton)

Insights into the
I
nteraction of beta
-
2
M
icroglobulin
F
ibrils with the
A
myloid
A
ssociated
P
rotein,
S
erum
A
myloid
-
P
C
omponent

17.35
-

18.00 Louise Butt

(Portsmouth University)

Collagen Recognition by Matrix
Metalloproteinase
-
1


18.00
-

19.15:

Time to check into rooms
.

University Arlott bar is open

19.15
-

20.00:

Drinks reception in Garden Court with live Jazz band

20.00 onward:

Conference dinner in Garden Court Hartley Suite

SWSBC 2012 University of Southampton


7

Tuesday July 17th 2012


08.00

-

09.0
0:

Breakfast
in
the
Terrace

restaurant (B38)
.
V
ouchers in
your
conference
pack


09.00
-

10.15
: Session 4
"Molecular Immunology"

09.00
-

09.2
5 Anna Fuller

(Cardiff University)

A Structural Analysis of T
-
Cell
E
scape by HIV

09.25
-

09.5
0 Chris Holland

(Cardiff University)

Dissecting the
B
inding
M
echanisms of a
C
ross
-
R
eactive, pMHC
C
lass II
R
estricted, T

C
ell
R
eceptor

9.5
0
-

10.
15

Andrea Schauenburg

(Cardiff University)

The
C
rystal
S
tructure of
L
atent and
I
mmunodominant Epstein
-
Barr
V
irus
-
D
erived T
C
ell Epitope


10.15

-

11.20:

Tea and coffee with sponsor exhibits and posters


11.20
-

12.50: Session 5
"Technology Advances"

11.20
-

11.45 Isabel Morares

(Imperial Col
lege London/ Diamond Light Source)

Challenges and Opportunities in Structure Determination of Membrane
Proteins

11.45
-

12.00 Paul Thaw

(Molecular Dimensions)

Crystallisation
S
trategies
-

from
C
lear
D
rops to
C
hampion
C
ryos

12.00
-

12.25 Ken Cunningham

(Avid

Nano)

Dynamic Light Scattering:

The State of the Art and Innovations to Look
Forward To

12.25
-

12.50 David Hall

(Diamond Light Source)

News from the MX
B
eam
L
ines at Diamond


12.50 onward

Lunch
(Terrace Restaurant B38)
and departure

















SWSBC 2012 University of Southampton


8

Additional
Events

on

Tuesday 17th afternoon


For those interested there will be tours of the new Southampton Diffraction Centre,
macromolecular crystallisation suite and the new Southampton BioNMR centre.


1)
Southampton

Diffraction Centre and Rigaku Europe Meeting


“Home X
-
ray sources and automation”



The Southampton Diffraction Centre (SDC) and Rigaku Europe are hosting a meeting
which is concerned with efficient use of home X
-
ray sources in the times of readily
availa
ble Synchrotron radiation: What is the advantage of in
-
house testing and data
collection, and what are the limits of in
-
house data acquisition?


With a combined small crystal and macromo
lecular set
-
up the Southampton
Diffraction Centre is uniquely suited
to discuss these questions. In running such a
facility, what is a healthy balance between collaborative academic projects and
service crystallography? The meeting also addresses techniques of macromolecular
crystallisation, the minimal required set
-
up and
the potential of the methods for crystal
detection and growth control.


The session is suitable for users of the equipment as well as those interested in setting
up this technology.


Programme:


14:00
-

14:05 Welcome

14:05
-

15:00 Panel discussion: What
capabilities do we expect from a home lab?

Panel Members include:

Simon Coles (SDC and National Crystallography Service)

Mark Benson

(Rigaku Europe)

Adam Irvine (Research &

Innovation Services, Southampton)

15:10
-

15:30 Tour I Diffraction; T
our II Crystallisation

15:10
-

15:30 Tour I Crystallisation; Tour II Diffraction

16:00
-

16:45 Coffee and round table discussion on:

(a) optimisation of the home lab and

(b) getting it right
-

the balance between commercial and academic

work.



Location: N
ightingale Building (67), Room 1003


2)
NMR Discussion Group


14.00
-
16.30

Discussion group regarding the regional NMR facilities initiative

Contributions are invited from the University of Oxford as the proposed centre and
representatives from
the Universities in the Southwest.


Location: Life Science Building (B85), room 2003

SWSBC 2012 University of Southampton


9

Sponsors


The meeting has been generously sponsored by the following companies and institutions
:





For more than 70 years, Agilent
Technologies has been the world's
premier measurement company.
Our singular focus on measurement
helps scientists and engineers
address their toughest challenges with precision and confidence. With the help of our
products a
nd services, they are better able to deliver the breakthroughs that make a
measurable difference in the world. Agilent Technologies X
-
ray group (formerly Oxford
Diffraction) design and manufacture single
-
crystal X
-
ray crystallography systems for structure
determination and high
-
throughput crystal diffraction screening.


Alpha Biotech is a distribution company with
market leading products for histocompatibility
(tissue typing for potential transplantation),
genetic screening, cell separation media,
protein

crystallography, nucleic acid
purification, sequence analysis software, BIOplastics, Brandel cell harvesters and
Suprafusion Systems service and maintenance.


Avid Nano are a UK based company specialising in
dynamic light scattering (DLS) systems for dem
anding
applications such as protein characterisation,
crystallography and purification. The technology
utilises a unique 5ul disposable measurement cuvette,
making the W130i DLS system an appealing
alternative to mainstream competition. Established in
2009
, Avid Nano's customer base already includes the likes of GSK, MRC Cambridge, Astex
Pharma, Sanofi Aventis and Novartis.



Bruker is a leading provider of high
-
performance scientific
instruments and solutions for molecular and materials research,
as well

as for industrial and applied analysis. Discover our
products and services.




Garland Science publish textbooks and multimedia
for the life sciences, biophysics, and chemistry, and
have been producing educational content of the
highest qu
ality for
nearly three decades.

We invite you to browse our website
www.garlandscience.c
om and find what interests you.
On the Products area of the site you
can find information on all of our books and multimedia, purchase printed texts, and also find

links to our e
-
book offerings.
In the Instructor and Student resource areas of the site, you can
search or browse all the multimedia designed to accompany our textbooks.

SWSBC 2012 University of Southampton


10


Leica Microsystems is a world leader in providing innovative
microscopy, camera and software soluti
ons for imaging and
analysis of macro
-
, micro
-

and nanostructures.




For over 30 years, Marresearch has been renowned
for the performance and reliability of their CCD and
imaging plate detector systems. These detectors can
be found on numerous synchrotro
n beam lines
worldwide as they meet the rigorous criteria demanded
by such facilities i.e. performance, reliability, data
quality, ease of operation etc. The detectors are used
in numerous different applications including PX, Small
Molecule, SAXS/WAXS, Mat
erials Science, High Energy Physics etc. Many home labs use
the Marresearch CCD and imaging plate detectors, usually in conjunction with the high quality
desktop beamline (dtb) goniometer system. Marresearch detector systems integrate with any
rotating ano
de or sealed tube/optic systems currently available.


Molecular Dimensions is a supplier of screens and
reagents for protein structure determination by x
-
ray
crystallography. We stock a wide variety of products for
protein crystal growth including the mar
ket leading
crystallization screens Morpheus and MIDAS,
crystallization plates (e.g. 96 well plates),
cryocrystallography and bacterial growth media. Molecular
Dimensions is the leading company for providing academic
-
led innovation in the crystallography c
onsumables market.

We also supply automated solutions for crystal imaging, UV fluorescence, and Dynamic Light
Scattering and are widely recognised as the world leader in membrane protein crystallization


The origins of the Oxford Cryosystems lie in the
design
and manufacture of the original Cryostream Cooler in
1985, which immediately became the system of choice for
cooling samples in X
-
ray diffraction experiments. The
range of products for use in sample cooling has
expanded over the last twenty
-
five yea
rs to include liquid
-
free systems, helium coolers and specially adapted
systems for use with powder samples. Today the
company is considered to be the global market leader in
X
-
ray diffraction sample cooling.


Palgrave Macmillan offers a wide range of Life Sciences
books and multimedia, published by W. H. Freeman, Sinauer
Associates, Inc. and University Science Books
-

all distributed
by Palgrave Macmillan
-

in addition to

books from Macmillan
Science and Palgrave Macmillan. At
www.palgrave.com/science you will find well
-
crafted
undergraduate and graduate textbooks that convey the excitement of scientific discovery,
professional books by authors working at the cutting
-
edge
of science and also popular
science books for the inquisitive reader. Many of our undergraduate textbooks are supported
by multimedia resources for lecturers and students.



Since its inception in Japan in 1951, Rigaku
Corporation has been at the forefro
nt of
analytical and industrial instrumentation
technology. With hundreds of major innovations
to its credit, Rigaku and its subsidiary
companies are world leaders in the fields of X
-
ray spectrometry, X
-
ray diffraction, X
-
ray optics,
as well as small molec
ule and protein crystallography.



SWSBC 2012 University of Southampton


11

Talk Abstracts


Thermophilic Enzymes and Applications in Commercial Biocatalysis

Jenny Littlechild


The Henry Wellcome Building for Biocatalysis, University of Exeter, Stocker Road, Exeter,

EX4 4QD, UK.


Naturally thermostable proteins are finding increasing applications in commercial biocatalysis.
In the Biocatalysis Centre in Exeter we have worked on several enzymes over the years;
some of which are already used for the production of pharmaceutical inter
mediates. These
enzymes form part of ‘white biotechnology’


a terminology used by the EU. Cloning, over
-
expression, biochemical and structural analysis of enzymes of interest can reveal factors
important for thermostability and substrate specificity.

Whe
n non
-
natural substrates are used different classes of enzyme can carry out the catalytic
turnover. This is the case for gamma lactam cleavage which can be carried out by a signature
amidase from the thermophilic archaeon,
Sulfolobus

species and a novel zi
nc enzyme related
to formamidase from the mesophile,
Comomonas

species. Both enzymes use different
mechanisms and have different structures. At non
-
physiological pH, side reactions can be
carried out which are different from the activity at pH 7.0. Another

gamma lactamase from
Aureobacterium
species with opposite stereoselectivity is a αβ hydrolase fold esterase but
can carry out a bromination reaction at pH 5.0.

Exeter is part of a large consortium grant called ‘Hotzyme’ where both metagenomics and
new nov
el thermophilic genomes are being used to screen for new thermostable enzymes
which can be identified from both bioinformatic and high throughput screening approaches.





SWSBC 2012 University of Southampton


12

Human Peroxiredoxin and Oxidative Stress

Paul James
, Misha Isupov and Jennifer Litt
lechild


Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental
sciences, University of Exeter, Stocker Road, Exeter EX4 4QD


Peroxiredoxins (Prx) are a family of multifunctional enzymes that protect against oxidative
stress and are produced in very high levels in cells. Prx form part of a peroxide scavenging
system along with Sulfiredoxin (Srx), Thioredoxin (Trx) and Thioredoxin reductase (TrxR). All
these enzymes use redox active cysteine residues to detoxify reactive

oxygen species or
reduce the other enzymes in the system.The structure of the red blood cell hPrxII was solved
in Exeter several years ago and showed a decameric structure with the peroxidatic cysteine
residue oxidised to sulfinic acid.

A new structure ha
s been solved that shows the decameric form of hPrxII in the disulfide state
that is known to form as part of its active cycle. This has revealed the necessary movements
that can occur to bring the peroxidatic and resolving cysteine residues together (prev
iously
~13Å apart) to form the disulfide bond. It was previously believed that the decameric form ofd
the protein could not exist in the disulfide state and only formed when the Prx was a dimer.

In addition an attempt was made to form the potential compl
exes between hPrxII and Trx, and
hPrxII and Srx. This was successful and has provided new information on the stoichiometry of
binding but to date diffraction quality crystals have not been obtained.





SWSBC 2012 University of Southampton


13

The Basis for Carbapenem Hydrolysis by Class A

-
L慣t
慭慳a猺s A 䍯浢楮敤
䥮I敳t楧慴楯i⁵獩 g⁃ 祳t慬aogr慰a礠ynd⁓業i污瑩lns

Fátima Fonseca
1,2
, Ewa I. Chudyk
1
, Marc W. van der Kamp
1
, António Correia
2
, Adrian J.
Mulholland
1

and
James Spencer
1


(1) University of Bristol

(2) University of Aveiro, Portugal


Carbapenems are the most potent

-
lactam antibiotics and key drugs for treating infections by
Gram
-
negative bacteria. In such organisms,

-
lactam resistance arises principally from

-
lactamase production. The class A

-
lactamases, the most prevalent of these enzymes, are
generally inhibited by carbapenems as the covalent acylenzyme intermediate deacylates very
slowly. However, carbapenem
-
hydrolyzing class A

-
lactamases are now disseminating in
clinically relevant bacte
ria. The reasons why carbapenems are substrates for some, but inhibit
other, class A

-
lactamases, remain to be fully established.

We have determined crystal structures of the class A carbapenemase SFC
-
1 from
Serratia
fonticola

and of two mutants that trap

the Michaelis and covalent acylenzyme complexes of
the carbapenem meropenem. The SFC
-
1 meropenem acylenzyme adopts an orientation
distinct from that observed in carbapenem
-
inhibited enzymes. Molecular dynamics
simulations indicate this mode of binding is
favoured by the wild
-
type Michaelis and
acylenzyme complexes. Structural comparisons show that subtle repositioning of key
residues (Ser70, Ser130, Asn132 and Asn170) enlarges the SFC
-
1 active site to permit this
reorientation of the carbapenem 6

-
1R
-
hydro
xyethyl group, preventing its interaction with the
water molecule responsible for deacylation and promoting efficient carbapenem hydrolysis.





SWSBC 2012 University of Southampton


14

The
Moraxella catarrhalis

DOXP R
eductoisomerase.

Richard Birkinshaw
, Leo Brady.


University of Bristol


The
2
-
C
-
methyl
-
D
-
erythritol 4
-
phospate (MEP) pathway produces isopentyl diphosphate (the
isoprenoid precursor) and is essential in many bacteria, algae, plants and protozoa. The MEP
pathway is replaced by the mevalonic acid pathway in humans, providing an att
ractive target for
developments of novel antimicrobials. The second enzyme in the MEP pathway is the 1
-
deoxy
-
D
-
xylulose 5
-
phosphate (DOXP) reductoisomerase (DXR), which catalyses rearrangement and
NAD(P)H dependent reduction of DOXP to MEP and is the targ
et of fosmidomycin, an
established antimicrobial and herbicide.

Moraxella catarrhalis

are gram
-
negative bacteria that are known to cause infection of the
respiratory tract, middle ear, eye, nervous system and joints in humans. With no approved
vaccine and

development of antibiotic resistant strains, there is a constant demand for innovative
therapeutics in the treatment of
M. catarrhalis

infections.

This talk presents 3 novel structures of the
M. catarrhalis

DXR (MDXR) in apo, ternary and
quaternary forms,

from which we can start to infer domain movements upon substrate binding.
The quaternary structure reveals a glycerol bound adjacent to the active site, which presents a
potential target for designing novel inhibitors. MDXR has been further characterise
d by enzyme
kinetics and FRET to unravel substrate and cofactor binding constants and investigate inhibitor
binding.





SWSBC 2012 University of Southampton


15

Structural Characterisation of the adhesion AspA, a key determinant of biofilm
formation in
Streptococcus pyogenes

Alice Robson,

Paul Race


University of Bristol


Streptococcus

species are usually commensal in the human oral cavity, but can cause
potentially life
-
threatening opportunistic infections. Cell
-
cell adhesion is a critical first step in
formation of bacterial biofilms necessary for invasion. The cell surface antigen As
pA from
S.
pyogenes

has been shown to have a key role in such mechanisms. It is a member of the
AgI/II family of adhesins, present in a number of
Streptococci
, which are characterised by
their long thin shape, extending ~50 nm from the cell. The AspA prote
in is relatively poorly
characterised and here we seek to rectify this using a combination of structural techniques.
We have expressed and purified the full
-
length protein (with the omission of the cell wall
anchor and signal sequence). It runs as a single

sharp peak on analytical gel filtration, with an
apparent molecular weight ~700kDa, despite the monomer weight of 140 kDa, indicative of
the elongated shape of the molecule. Ongoing studies on the full
-
length protein by electron
microscopy and on various
individual domains using x
-
ray crystallography will be described.





SWSBC 2012 University of Southampton


16

Decoding the biosynthesis of the antibiotic Mupirocin
-

the role of MupS

Marisa Till
, Paul Race.


Department of Biochemistry, University of Bristol.


The broad spectrum antibiotic mupirocin is used clinically in the prevention and treatment of
bacterial infections in burns and skin graft patients. This compound, a member of the
polyketide class of natural products, is produced by the bacterium
Pseudomon
as
fluorescence
, and shows significant bactericidal activity against MRSA. The inherent chemical
instability of mupirocin has to date restricted its use to topical application, however, there is
significant potential for modification of the chemical scaffo
ld of this molecule towards the
development of more clinically viable "mupirologues". A prerequisite for achieving this goal is
the provision of a detailed structural and functional description of the mupirocin biosynthetic
pathway.

The ~ 70 kb gene cluste
r encoding the machinery responsible for the biosynthesis of
mupirocin has been identified and sequenced. In a collaborative effort between the
Universities of Bristol and Birmingham we are currently investigating the roles of the individual
components of
this pathway. Here I will describe our recent progress in the structural and
functional characterisation of one of the mupirocin pathway enzyme MupS, which has been
implicated to play a role in the generation of a highly unusual 3
-
Hydroxy propanate starter

unit.





SWSBC 2012 University of Southampton


17

LcrH, a Class II C
haperone fro
m Type Three Secretion System, H
as a
Highly Flexible
Native S
tructure.

Sunny Singh
1, 2
, Aimee Boyle
3

& Ewan Main
1
.


(1)

Queen Mary University of London

(2)

University of Sussex

(3)

University of Bristol


Many Gram
-
negative
pathogens employ a large complex nano
-
machine called Type Three
Secretion System (T3SS) to infect host eukaryotic cells. The key structure of this machine is
a proteinaceous pore that inserts into the target membrane and forms a channel for passage
of bac
terial toxins. The pore is formed mainly by two large membrane proteins
(‘’translocators’’) which are transported from the bacterial cytosol to the membrane by small
specialised chaperones.

Recently crystal structures have revealed that these chaperones a
re made of modular
tetratricopeptide repeats. We investigated the conformational stability of the chaperone LcrH
(
Yersinia pestis)
.
Circular dichroism monitored equilibrium denaturations, analytical
ultracentrifugation and size exclusion chromatography

sho
wed that LcrH is a weak and
thermodynamically stable dimer (
K
D

≈ 15 µM, ΔG
H2O

= 7.4 kcal.mol
-
1
). Modular TPR structure
of LcrH allows it to readily unfold in a non
-
cooperative manner to a one
-
third unfolded dimeric
intermediate (ΔG
H2O

= 1.7 kcal.mol
-
1
), be
fore cooperatively unfolding to a monomeric
denatured state (ΔG
H2O

= 5.7 kcal.mol
-
1
). Thus, under physiological conditions LcrH is able to
populate C
-
terminally unravelled partially folded states, whilst being held together by its
dimeric interface. Such a
bility suggests a ‘’fly
-
casting’’ mechanism as a route to binding their
far larger translocator cargo.





SWSBC 2012 University of Southampton


18

Computational
and expression studies of the
Chlamydia pneumoniae

Major Outer
Membrane Protein (MOMP)

Francis O Atanu
, Ian M Jones and Kimberly A
Watson


School of Biological Sciences, University of Reading, UK


The Major Outer Membrane Protein (MOMP) of the genus
Chlamydia

is a cysteine rich 40
kDa protein localised to the outer membrane.
C. pneumoniae
is a human patho
gen
responsible for at least
20
% of lower respiratory tract infections. Its role in the development of
atherosclerosis has been justified by several lines of evidence. Recently, the anti
-
atherosclerotic activities and modulation of cell mediated immunity by recombinant MOMP
from
C. pn
eumoniae

was demonstrated.

In this report, we present a three dimensional homology model of MOMP based on the fatty
acid transporter of
E. coli

and the most plausible knowledge based assignment of function
based on orientation of identified motifs. We tested the hypothesis of cell mediated
modulation of immunity by preferentially docking four MOMP derived peptides unto Major
Histocompatibility Co
mplex (MHC) class II.

Our results were corroborated by predictions for immunoinformatic applications. Expression
and purification studies of MOMP suggest the likelihood to localise to the inner membrane
when expressed in non
-
Chlamydial

host and purifiable

at ~100 µg per liter.





SWSBC 2012 University of Southampton


19

Macromolecular Applications with the New, Brighter Nova X
-
ray Source from Agilent
Technologies

Marcus J. Winter


Agilent Technologies, Oxford Industrial Park, Yarnton, Oxfordshire, OX5 1QU, UK


Agilent Technologies develops and
supplies a range of instruments for a variety of single
-
crystal X
-
ray diffraction applications. Notable are the SuperNova: a compact, highly reliable
and very low maintenance system providing complete X
-
ray data of the highest quality, and
the PX Scanner f
or the testing and

characterisation

of protein crystals in their original

crystallisation

drops (
in
-
situ
).

Recently we have incorporated a number of enhancements to both instruments to make them
even more powerful and effective. The principal change invol
ves the introduction of the
second generation of the Nova (Cu) microfocus sealed
-
tube X
-
ray source: this results in over
2x stronger diffraction from protein crystals, with a consequent significant improvement in
signal
-
to
-
noise.

We present results of rece
nt experiments carried out on both the SuperNova and PX Scanner
which demonstrate that

with these new instruments Agilent provides highly effective X
-
ray
solutions for the structural biology community which perfectly complement the usage of
synchrotron bea
m lines.





SWSBC 2012 University of Southampton


20

Insights into the interaction of beta
-
2 microglobulin fibrils with the amyloid associated
protein, serum amyloid
-
P component

Garrick Foster Taylor
1

, Steve Wood
2
, Jörn Werner
1

and Philip Williamson
1


(1)

School of Biological Sciences, University of Southampton, University Rd, Southampton,
SO17 1BJ United Kingdom

(2)
Centre of Amyloidosis and Acute Phase Proteins, Royal Free and University College
Medical School, Rowland Hill St, London, NW3 2PF, United K
ingdom

(

)
Current address: Department of Biochemistry, University of Oxford, South Parks Rd,
Oxford, OX1 3QU, United Kingdom


Dialysis related amyloidosis (DRA) results in the deposition of amyloid in the joints causing
pain and restricted mobility for su
fferers. The major protein component of these amyloid
deposits is fibrillar β2
-
microglobulin (β2m). Serum amyloid
-
P component (SAP) is a protein
ubiquitously present in fibrillar deposits and is thought to play a key role in stabilising the
fibrillar struc
tures and preventing their clearance by the host’s defences.

Using solid and solution
-
state NMR we investigated the structural transitions that result in the
conversion of monomeric β2m into its fibrillar form and identified sites involved in the
interact
ion with SAP. Magic
-
angle spinning (MAS) correlation experiments have permitted the
substantial assignment of the fibrils. PDSD experiments of labelled fibrils in the presence of
unlabelled SAP reveal significant changes in the spectral region correspondin
g to the fibril’s
glutamate sidechains. Modification of the glutamate sidechains of amyloid fibrils to remove
their charge results in complete loss of binding between the fibrils and SAP, highlighting the
essential nature of the fibril glutamate sidechains

for binding with SAP. Saturation transfer
difference experiments have revealed that glutamates compete for the calcium binding site of
SAP, thereby providing a plausible and blockable site of interaction between amyloid fibrils
and SAP.





SWSBC 2012 University of Southampton


21

Collagen
Recognition by Matrix Metalloproteinase
-
1

Louise Butt

and Andy Pickford


University of Portsmouth


Matrix Metalloproteinase
-
1 (MMP
-
1) is an instigator of triple helical collagen catabolism. The
mechanism of collagen recognition and cleavage by
collagenases remains poorly understood.
Previous crystallographic studies have revealed that proMMP
-
1 activation


by removal of its
inhibitory propeptide

domain
-

is accompanied by structural rearrangements between the
catalytic
and hemopexi
n
domain, resulting in an “open” conformation. It is believed that this
conformational change reveals a putative binding site in the mature MMP
-
1 enzyme, a
characteristic that is reportedly abs
ent from the proMMP
-
1 zymogen.

This study uses site
-
dire
cted mutagenesis
, surface plasmon resonance

and small
-
angle X
-
ray
scattering
to investigate the contribution of interdomain interactions to the “closed”
conformation of proMMP
-
1 and hence to the modulation of its collagen binding
activity.




SWSBC 2012 University of Southampton


22

A Structural Analysis of T
-
Cell escape by HIV

Anna Fuller
, David Cole, Pierre Rizkallah, Andrew Sewell.


Cardiff University


Human immunodeficiency virus (HIV) can infect and eliminate CD4+ T
-
cells. Depletion of
these cells, that form an impo
rtant wing of the adaptive cellular immune system, is a major
factor leading to acquired immune deficiency syndrome (AIDS). CD8+ T
-
cells have the ability
to eliminate cells infected with HIV and other viruses through an interaction between the T
-
cell
recep
tor (TCR) and peptide
-
major histocompatiblity complex class

I (pMHCI) at the cell
surface.

However, HIV can rapidly mutate many of its protein components to avoid detection by
circulating CD8+ T
-
cells. The HLA A*0201 restricted HIV
-
GAG epitope (SLYNTVATL)
(A2
-
SLY) has been shown to be a major target for CD8+ T
-
cell immune responses. A number of
escape variants derived from this epitope have been extensively investigated. In order to
study the structural basis of HIV immune escape based on this epitope, we h
ave solved the
structure of the a TCR (868 TCR), derived from an A2
-
SLY specific CD8+ T
-
cell clone (868),
alone and in complex with wildtype A2
-
SLY, and 2 well characterized escape mutant variants
(A2
-
SLY
6I
; SLYNT
I
ALT and A2
-
SLY
3F6I8V
; SL
F
NT
I
A
V
T). These
novel data provide
important insights into the alterations in the structural elements of the A2
-
SLY variants during
binding to the 868 TCR that lead to evasion of CD8+ Tcell mediated immunity by HIV.





SWSBC 2012 University of Southampton


23

Dissecting the binding mechanisms of a
cross
-
reactive, pMHC class II restricted, T cell
receptor

Chris Holland
, Pierre Rizkallah, David Cole
.


Cardiff University


Successful immunity requires that a limited pool of αβ T
-
cell receptors (TCRs) provide cover
for a vast number of potential foreign peptide antigens presented by ‘self’ major
histocompatibility complex (pMHC) molecules. Structures of unligated and ligated
MHC class
-
I
-
restricted TCRs with different ligands have revealed a number of important mechanisms that
govern TCR mediated antigen recognition. However, due to a lack of structural data, the
mechanisms that govern recognition by MHC class
-
II
-
restricted TCR
s are unclear.

HA1.7 TCR binding to the influenza hemagglutinin antigen (HA306
-
318) presented by HLA
-
DR1 and HLA
-
DR4 represents a good system for interrogating the molecular rearrangements
involved in accommodating pMHC
-
II ligands. Accordingly, we solved
the structure of the
unligated HA1.7 TCR and compared it to both complex structures. The HA1.7 TCR underwent
minimal conformational adjustments during ligand engagement. Despite this relatively rigid
binding mode, HA1.7 T
-
cells could tolerate some mutation
s in key contact residues within the
peptide epitope. Thermodynamic investigations revealed the HA1.7 TCR undergoes the
largest enthalpic cost so far reported for a TCR
-
pMHC interaction. Thus, limited plasticity and
favourable entropy underpin the ability
of the HA1.7 T
-
cell clone to crossreact with multiple
MHC
-
II alleles and different antigenic peptide.





SWSBC 2012 University of Southampton


24

The crystal structure of latent and immunodominant Epstein
-
Barr virus
-
derived T cell
Epitope

Andrea J. Schauenburg
, John J. Miles, David K. Cole, Joha
nne Pentier, Emma Gostick, Linda
Wooldridge, Pierre J. Rizkallah, Andrew K. Sewell and David A. Price.


Cardiff University
,
Cardiff
,
Wales
.
CF10 3XQ
.
UK


Epstein
-
Barr virus (EBV) is a flourishing microparasite presently infecting over 90% of the
human population. The virus is also a global disease burden and associated with 18 different
malignancies. The latent EBV protein LMP2 is a key target of the cellul
ar immune system
since it encodes a number HLA
-
A*0201 (A2) restricted epitopes including FLY (FLYALALLL).

FLY is a promising therapeutic and prophylactic target since it is expressed on the surface of
most EBV
+

malignancies and exhibits complete sequence
conservation across all known virus
strains. How T cells engage this important epitope is unknown. Here, we have solved the
structure of the A2
-
FLY molecule in complex with the public TCR SB7 and have identified key
areas of T cell contact.

Understanding
the molecular basis of this engagement may aid in optimizing T cell
recognition as well as the design of intelligent CLG mimics compounds and super
-
agonists for
use in the clinic.





SWSBC 2012 University of Southampton


25

Challenges and Opportunities in Structure Determination of Membrane
Proteins

Isabel Moraes


Membrane Protein Laboratory
,
Diamond Light Source Ltd
,

Chilton, Didcot, Oxfordshire,

OX11 ODE


Membrane proteins are the supreme example where more effort is needed in structural
biology. In spite of their abundance and importance,

of the more than 60,000 protein
structures in the Protein Data Bank, only around 200 of these structures represent unique
membrane proteins.

To facilitate structural studies on membrane proteins, The Membrane Protein Laboratory at
Diamond Light Source Ltd

(Diamond
-
MPL) was created in a collaboration between Imperial
College London and Diamond, funded by the Wellcome Trust.

The MPL is a research and training state
-
of
-
the
-
art user facility open to scientists from
laboratories anywhere in the world interested

in solving the 3
-
dimensional structures of
membrane proteins by X
-
ray crystallography.
Because membrane proteins are unstable, hard
to crystallise and crystals difficult to handle. More systematic approaches and technical
developments are needed to improv
e the success rate of the structure determination of
membrane proteins.

The MPL has a formal collaboration with I24 microfocus beamline at Diamond to develop new
techniques for crystallisation and structural determination of membrane proteins, including
pr
ototype systems for high throughput methods, improving handling of small and delicate
crystals, and methods for collecting and merging data from a large number of small crystals.

The beamline is unique in its ability to deliver a tunable X
-
ray beam (6


25
keV) of variable
size between 5 µm and 50 µm on to crystal samples. This versatility is coupled with a state
-
of
-
the
-
art pixel array detector.







SWSBC 2012 University of Southampton


26

Crystallisation strategies
-

from clear drops to champion cryos.

Paul Thaw


Molecular Dimensions


Traditionally protein crystallography has been a fairly stochastic affair with "dumb luck" and
sheer determination been major factors in success. In an ideal world the protein would always
be pure, crystallize by lunchtime and the structure finished that d
ay. Dreaming apart, the
closest we can currently get to this is to reduce the pain by minimizing the time, effort and
money taken to get the result we crave. A thoughtful, informed approach to crystallization can
be as productive if not more so than the "k
itchen sink" approach
-

high output is always
preferable to high throughput.

A willingness to combine economical screening with informative biophysics reduces protein
expenditure and can guide crystallization even during the process itself. Speedier and si
mpler
identification of every crystallization lead and careful attention to freezing the crystal should
ensure good quality data collection. The talk will address our latest thinking in these areas
and hopefully stimulate some discussion on streamlining th
e crystallization pipeline in the face
of increasingly more difficult targets.





SWSBC 2012 University of Southampton


27

Dynamic Light Scattering:

The State of the Art and Innovations to Look Forward To.

Ken Cunningham


Avid Nano Ltd


Ken Cunningham is the founder and owner of Avid Nano
Limited, a UK based company
specialising in dynamic light scattering (DLS) systems for demanding applications like protein
cry
stallography and purification.

The technology is protected by a patent application for a low volume disposable
measurement cuvett
e, making the W130i DLS system a uniquely appealing alternative to
mainstream competition. In this presentation, Ken will assess the current state of the art, its
advantages and limitations. He also looks at the development pipeline to see where DLS is
goi
ng and how it can offer even more to research in the nano arena.





SWSBC 2012 University of Southampton


28

News from the MX beam lines at Diamond

David Hall


Diamond Light Source


Diamond Light Source supports UK (and worldwide) structural biologists by providing
beamlines dedicated to life sc
iences. In particular in support of the internationally competitive
UK macromolecular crystallography (MX) community it provides a suite of MX beamlines.

There are currently five fully operational beamlines (I02, I03, I04, I04
-
1 and I24) which will be
joi
ned in the not too distant future by a long wavelength beamline, I23. More recently support
was given for the development of two new end
-
stations dedicated to sub
-
micron focus and
in
-
situ

diffraction experiments respectively. This talk will review the status of the available and
future beamlines for MX at Diamond Light Source.












SWSBC 2012 University of Southampton


29

Poster Abstracts


1)
Insights into the interaction of beta
-
2 microglobulin fibrils with the amyloid
associated protein, serum amyloid
-
P component

Garrick Foster Taylor
1

, Steve Wood
2
, Jörn Werner
1

and Philip Williamson
1


(1)
School of Biological Sciences, University of Southampton, University Rd, Southampton,
SO17 1BJ United Kingdom

(2)
Centre of Amyloidosis and Acute Phase Proteins, Royal Free and University College
Medical School, Rowland Hill St, London, NW3 2PF, United Ki
ngdom

(

)
Current address: Department of Biochemistry, University of Oxford, South Parks Rd,
Oxford, OX1 3QU, United Kingdom


Dialysis related amyloidosis (DRA) results in the deposition of amyloid in the joints causing
pain and restricted mobility for suf
ferers. The major protein component of these amyloid
deposits is fibrillar β2
-
microglobulin (β2m). Serum amyloid
-
P component (SAP) is a protein
ubiquitously present in fibrillar deposits and is thought to play a key role in stabilising the
fibrillar struct
ures and preventing their clearance by the host’s defences.

Using solid and solution
-
state NMR we investigated the structural transitions that result in the
conversion of monomeric β2m into its fibrillar form and identified sites involved in the
interacti
on with SAP. Magic
-
angle spinning (MAS) correlation experiments have permitted the
substantial assignment of the fibrils. PDSD experiments of labelled fibrils in the presence of
unlabelled SAP reveal significant changes in the spectral region corresponding

to the fibril’s
glutamate sidechains. Modification of the glutamate sidechains of amyloid fibrils to remove
their charge results in complete loss of binding between the fibrils and SAP, highlighting the
essential nature of the fibril glutamate sidechains
for binding with SAP. Saturation transfer
difference experiments have revealed that glutamates compete for the calcium binding site of
SAP, thereby providing a plausible and blockable site of interaction between amyloid fibrils
and SAP.






2)
Structural and functional studies of a
Streptococcus suis

surface protein

Rosamund Ellis
1
, Chris Williams
1
, Howard F. Jenkinson
2
, Matthew P. Crump
1


(1)
School of Chemistry, University of Bristol

(2)
School of Oral and Dental Sciences, University of
Bristol


The Uncovering Enzyme (UCE) is a human protein involved in the formation of a recognition
tag which facilitates transport of lysosomal enzymes to the lysosomes. Incorrect formation of
this tag can lead to lysosomal storage diseases.

Previously a
sample of the catalytic domain of this human protein proved difficult to purify,
however there are various bacterial analogues of UCE which are easier to express using an
E. coli

expression system. One of these proteins is a potential exopolysaccharide
bi
osynthesis protein from
Streptococcus suis
, an important zoonotic pathogen.

The catalytic domain of this bacterial protein has been expressed and purified and high
quality NMR spectra were recorded on
13
C
15
N and
2
H
13
C
15
N labelled samples. Spectral
assign
ment is ongoing with the aim of structure determination.

The function of the
S. suis

UCE analogue and related proteins has also been investigated by
in vivo assays, as the absence of lysosomes in prokaryotes indicates a different role for this
bacterial protein.




SWSBC 2012 University of Southampton


30

3)
Structure of a novel phosphotyrosine
-
binding domain in Hakai that
targets E
-
cadherin.

Manjeet Mukherjee
, Soah Yee Chow, Permeen Yusoff, J Seetharaman, Cherlyn Ng,
Saravanan Sinniah, Xiao Woon Koh, Nur Farehan M Asgar, Dan Li, Daniel Yim, Rebecca A
Jackson, Jingxi Yew, Jingru Qian, Audrey Iyu, Yoon Pin Lim, Xingding Zhou,

Siu Kwan Sze,
Graeme R Guy and J Sivaraman


National University of Singapore, Singapore


Phosphotyrosine
-
binding domains, typified by the SH2 (Src homology 2) and PTB domains,
are critical upstream components of signal transduction pathways. The E3
ubiquitin ligase
Hakai targets tyrosine
-
phosphorylated E
-
cadherin via an uncharacterized domain. In this
study, the crystal structure of Hakai (amino acids 106

206) revealed that it forms an atypical,
zinc coordinated homodimer by utilizing residues from t
he phosphotyrosine
-
binding domain of
two Hakai monomers. Hakai dimerization allows the formation of a phosphotyrosine
-
binding
pocket that recognizes specific phosphorylated tyrosines and flanking acidic amino acids of
Src substrates, such as E
-

cadherin, cor
tactin and DOK1. NMR and mutational analysis
identified the Hakai residues required for target binding within the binding pocket, now named
the HYB domain. ZNF645 also possesses a HYB domain but demonstrates different target
specificities. The HYB domain is
structurally different from other phosphotyrosine
-
binding
domains and is a potential drug target due to its novel structural features.










4)
The crystal structure of latent and immunodominant Epstein
-
Barr virus
-
derived T
cell Epitope

Andrea J.
Schauenburg
, John J. Miles, David K. Cole, Johanne Pentier, Emma Gostick, Linda
Wooldridge, Pierre J. Rizkallah, Andrew K. Sewell and David A. Price.


Cardiff University
,
Cardiff
,
Wales
.
CF10 3XQ
.
UK


Epstein
-
Barr virus (EBV) is a flourishing microparasite presently infecting over 90% of the
human population. The virus is also a global disease burden and associated with 18 different
malignancies. The latent EBV protein LMP2 is a key target of the cellul
ar immune system
since it encodes a number HLA
-
A*0201 (A2) restricted epitopes including FLY (FLYALALLL).

FLY is a promising therapeutic and prophylactic target since it is expressed on the surface of
most EBV
+

malignancies and exhibits complete sequence conservation across all known virus
strains. How T cells engage this important epitope is unknown. Here, we have solved the
structure of the A2
-
FLY molecule in complex with the public TCR SB7 and have identified

key
areas of T cell contact.

Understanding the molecular basis of this engagement may aid in optimizing T cell
recognition as well as the design of intelligent CLG mimics compounds and super
-
agonists for
use in the clinic.









SWSBC 2012 University of Southampton


31

5)
Crystallisation and
Structural Studies on Omega Transaminase Enzymes

Aaron Westlake,

Dr Christopher Sayer, Dr
Michail Isupov, Professor Jennifer Littlechild
.


University of Exeter


Omega transaminase enzymes are proving to be of vital importance in the production of chiral
amines for drug manufacturing due to their high stereoselectivity and regioselectivity
reactions. Omega transaminases also possess another key feature that can mak
e them more
desirable than other transaminase enzymes in that they are able to utilise substrates lacking a
carboxyl group.

This project has purified several recombinant omega transaminases from the bacteria;
Streptomyces avermitilis
,

Pseudomonas putida

a
nd
Pseudomonas aeruginosa
. Attempts have
been made to crystallise the proteins in order to determine their structures. Co
-
crystallisation
experiments with
P. aeruginosa

omega transaminase and the inhibitor gabaculine were
successful.

Data collected from a
Chromobacterium violaceum

omega transaminase with a F89 to A89
mutation was successfully processed and refined. The resultant structure showed an increase
in flexibility in the substrate carboxyl binding residue, R416, and an increase in space
available fo
r substrate binding. HPLC assays showed alpha
-
methylbenzylamine activity
remained. An amino acid oxidase/horseradish peroxidase linked assay showed there to be a
small increase in activity towards
β
-
alanine due to this mutation. Further mutations will be
n
ecessary to enable this enzyme to utilise
β
-
alanine at a more practical rate of conversion.











6)
Structural Characterisation of the adhesion AspA, a key determinant of biofilm
formation in
Streptococcus pyogenes

Alice Robson,

Paul Race


University
of Bristol


Streptococcus

species are usually commensal in the human oral cavity, but can cause
potentially life
-
threatening opportunistic infections. Cell
-
cell adhesion is a critical first step in
formation of bacterial biofilms necessary for invasion. Th
e cell surface antigen AspA from
S.
pyogenes

has been shown to have a key role in such mechanisms. It is a member of the
AgI/II family of adhesins, present in a number of
Streptococci
, which are characterised by
their long thin shape, extending ~50 nm from

the cell. The AspA protein is relatively poorly
characterised and here we seek to rectify this using a combination of structural techniques.
We have expressed and purified the full
-
length protein (with the omission of the cell wall
anchor and signal seque
nce). It runs as a single sharp peak on analytical gel filtration, with an
apparent molecular weight ~700kDa, despite the monomer weight of 140 kDa, indicative of
the elongated shape of the molecule. Ongoing studies on the full
-
length protein by electron
m
icroscopy and on various individual domains using x
-
ray crystallography will be described.









SWSBC 2012 University of Southampton


32

7)
Determining the role of NS4B membrane remodelling in HCV replication

Esther von Schulthess Rechberg
1
, Chris McCormick
2

and Philip T. F. Williamson
1



(1) Centre for Biological Sciences, Highfield Campus, University
of Southampton,
Southampton, UK

(2) School of Medicine, Southampton General Hospital, University of Southampton,
Southampton, UK.


Hepatitis C is an RNA virus that replicates in association with intracellular membranous
structures call membranous webs (MWs). Viral protein NS4B is a key organizer of replication,
one crucial function being the induction of MWs. The mechanisms of MW form
ation are
unknown, but it clearly involves induction of membrane curvature, which may require NS4B
oligomerisation and possibly hydrophobic wedging. NS4B is known to oligomerise, and the N
-
terminal amphipathic helix AH2 has been implied as a major determin
ant of self
-
association.
In order to understand the process of MW induction, we aimed to determine AH2’s capacity to
remodel membranes by studying the interaction of AH2 with membranes mimicking those
found within the cell using 2H and 31P solid
-
state NMR.

Our results show changes in
membrane morphology, induced by AH2 in negatively charged vesicles, an effect not
observed in neutral bilayers indicating a requirement for negatively charged lipids. Chemical
cross
-
linking studies of AH2 in lipids vesicles con
firms AH2 homo
-
oligomerisation and
suggests a charge dependency; with larger oligomers observed in neutral lipid bilayers
compared to negatively charged lipid bilayers and lipid mixtures mimicking cellular
membranes. These results suggest that AH2 plays a
crucial role in NS4B’s capacity to alter
membrane morphology.






8)
Improved solid
-
state NMR methods for the analysis of nitrogen
-
14 in biosolids

James Jarvis
1
, Ibraheem Haies
2
, Marina Carravetta
2
,
Philip T.F. Williamson
1


(1) Centre for Biological
Sciences, Highfield Campus, University o
f Southampton,
Southampton, UK.

(2) School of Chemistry, Highfield Campus, University of Southampton, Southampton, UK.


Nitrogen is one of the most abundant elements and plays a key role in the chemistry of
biologica
l systems. Despite its widespread distribution the study of the naturally occurring
isotope of nitrogen,
14
N (99.6%), has been relatively limited as it is a spin
-
1 nucleus that
typically exhibits a large (>MHz) quadrupolar interaction. Accordingly most stu
dies of nitrogen
sites in biomolecules have been performed on samples enriched with
15
N, limiting the
application of NMR to samples which can be isotopically enriched. This precludes the analysis
of naturally occurring samples and results in the loss of th
e wealth of structural and dynamic
information that the quadrupolar interaction can provide.

Recently experiments have been developed which permit the characterisation of
14
N sites
through their interaction with neighbouring ‘spy’ nuclei (reviewed in Cavad
ini, Prog. NMR
Spec. 2010
56
, 46
-
77). Here a novel version of these experiments is described whereby
recoupling of the interactions between the
14
N site and the spy nucleus is mediated by the
application of a moderate rf field to the
14
N. The resulting
13
C
/
14
N spectra show good
sensitivity on natural abundance (25% vs CP) and labelled materials (15% vs CP); whilst the
14
N lineshapes can provide a quantitative analysis of the quadrupolar interaction. Using this
approach we are using the information encoded i
n the quadrupolar interaction to study the
structure and dynamics of biomolecules.







SWSBC 2012 University of Southampton


33

9)
Structure, function and role of the hERG pore helix in the long QT syndrome
-

an
NMR study


Maïwenn Beaugrand
1
, Alexandre A. Arnold
1
, Philip T. F. Williamson
2

and Isabelle Marcotte
1


(1) Department of Chemistry, Université du Québec à Montréal, Montreal (Québec) Canada.
(2) Centre for Biological Sciences, Highfield Campus, University of Southampton,
Southampton, UK.


The long QT syndrome (LQTS) is a cardiac dysfunction that prolongs the heart repolarisation
interval, leading to cardiac arrhythmia or failure. This pathology can be induced by off target
effects of drugs which block the human ether
-
a
-
go
-
go related gene (h
ERG) potassium
channels located in the myocardium cell membranes. To reduce the risks of this acquire
LQTS (ALQTS), regulatory authorities demand
in vitro

testing of all new drug entities for
hERG
-
blocking potential. The problem of ALQTS is correlated with

the wide spectrum of
hERG blockers which are structurally diverse and from different pharmacological classes.
Because the pore domain of the hERG channel is an important target of LQTS
-
prone drugs,
the aim of our work is to investigate the function and ro
le of the pore helix (P
H
) in the ALQTS
using a combined solution and solid
-
state (SS) NMR approach. We have first studied the
interaction of the amphiphilic P
H

(G
603
-
G
626
) with the membrane. Using bicelles as model
membranes, our
31
P and
2
H SS
-
NMR results
show that the P
H
induces the formation of
isotropic and oriented lipid phases. Moreover, an increased lipid chain ordering is observed in
the oriented phase in the presence of the P
H

especially in the plateau region of the bilayer
where the peptide would b
e located. The interaction of the P
H

with LQTS
-
active drugs was
studied by solution NMR using translational diffusion and saturation transfer difference
experiments. The drug affinity and interaction sites will be discussed.









10)
Struc
ture And Opera
tion Of Type I DNA

Restriction/Modification Enzymes

Anna Swiderska
, James Taylor, Christopher Kennaway, Geoff Kneale


Portsmouth University, PO1 2DY


Type I restriction/modification (RM) enzymes are found in all known bacteria. They are
comprised of two,
large multi
-
subunit enzymes: the methyltransferase, responsible for site
-
specific methylation (and thus protection) of the host DNA; and the restriction endonuclease,
responsible for the cleavage of foreign, unmethylated DNA. Both enzymes share a number of

subunits. The endonuclease reaction is preceded by bidirectional translocation of thousands
of base pairs of DNA in an ATP dependent mechanism.

Crystallisation of these large multi
-
subunit enzymes has been unsuccessful, despite the
efforts of many laborat
ories. Only a few crystal structures, mainly of partial subunits, have
been solved and the overall architecture of the complex is unknown. Here we present the
structures of two type I RM enzymes, obtained by using combination of different techniques:
elect
ron microscopy (EM), small
-
angle scattering (neutron and x
-
ray), and molecular
modelling. Binding of DNA induces a conformational change in the enzymes


a shift from an
open to a closed form. The path followed by DNA through the complexes is revealed by u
sing
a DNA mimic, anti
-
restriction protein [1].

[1]
Genes Dev.2012, Jan 1; 26(1):92
-
104







SWSBC 2012 University of Southampton


34

11)
Structural Characterisation of the first animal Cellobiohydrolayse

J. McGeehan
1
, S. Streeter
1
, R. Martin
1
, M. Kern
2
, N. Bruce
2
, G. Beckham
3
, C. Payne
3
, M.
Himmel
3

& S. Cragg
1


(1) School of Biological Sciences, Universi
ty of Portsmouth, PO1 2DY UK

(2) Department of Biology, University of Y
ork, Heslington, York, YO10 5DD

(3) NREL, Golden, Colorado, USA


The GH7 family of enzymes are ubiquitous in wood digesti
ng fungi and have gained a great
deal of attention over recent years due their key role in the metabolism of cellulose to simple
and bio
-
available sugars. These enzymes deliver the highest known hydrolytic potential on a
per mole basis compared to any othe
r know cellulase family and therefore represent an
important engineering target for biofuels production.

Following on from the exciting discovery that a subset of marine borers and related species
contain novel GH7 enzymes
1
, we have structurally character
ised the GH7B from
Limnoria
quadripunctata
. This is the first animal GH7 to be solved by X
-
ray crystallography and we
reveal that it is both evolutionarily and functionally divergent from the non
-
animal
homologues.

Analysis of the free and substrate
-
bound

structures reveal details of the active site tunnel and
the flexible loop regions that together bestow substrate specificity and activity, while molecular
dynamics simulations illustrate how this enzyme binds and processes cellulose. These
detailed studie
s will directly inform protein engineers who aim to generate novel chimeric
Limnoria
-
fungal enzymes.


[
1
]

King AJ, Cragg SM, Li Y, Dymond J, Guille MJ, Bowles DJ, Bruce NC, Graham IA,
McQueen
-
Mason SJ. (2010)
PNAS

23, 107(12), 5345
-
50



12)
Biophysical
studies of a novel controller protein and its interaction with DNA.



Mikhail Shevtsov
, John McGeehan, Simon Streeter, Sara
h
-
Jane Thresh and Geoff Kneale


IBBS, University of Portsmouth, PO1 2DT, UK


In a wide variety of bacterial restriction
-
modification
systems, a regulator protein, the C
-
protein, is required for effective transcription of its own gene, and for transcription of the
endonuclease (R) gene found on the same operon (1,2)
. In our work we study a new class of
controller protein (exemplified by
C.Csp231I) that has some novel features. In particular, it
has an
extended C
-
terminal region that is absent in all other known C
-
protein structures and a
long spacer (18 b.p.) between the two palindromic DNA binding sequences (3). We have
studied the bindi
ng of C.Csp with its target DNA sequence employing variety of structural and
functional techniques in order to define the genetic switch that governs the time
-
dependence
of controller protein (and thus endonuclease) expression. Our data suggest a novel str
ucture
of the “spacer” DNA separating the two C
-
protein dimer binding sites.



[1] Tao T, Bourne
et al
.
(1991)

A family of regulatory genes associated with type II restriction
-
modification systems
. J. Bacteriol;
173
,

1367
-
1375.

[2] McGeehan JE,
et al
.
(2008) Structural analysis of the genetic switch that regulates the
expression of restriction
-
modification genes.

Nucleic Acids Res;
36
,

4778
-
4787.

[3] McGeehan JE,
et al
.
(
2011
)
Structural analysis of a novel class of R
-
M controller proteins:
C.Csp231I from
Citrobacter sp. RFL231
.
J Mol Biol
;
409
,

177
-
188.


SWSBC 2012 University of Southampton


35

13)
The Molecular Basis of Protein
-
DNA Recognition in a Bacterial Genetic Switch

R.N.A. Martin
, N.J. Ball, J.E. McGeehan & G.
G. Kneale


Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, UK


The bacterial protein
C.Esp1396I

is a helix
-
turn
-
helix (HTH) transcription factor that controls
the expression of both a methyltransferase and endonuclease in a type II restriction
-
modification system.
C.Esp1396I

achieves this by interacting with three subtly different DNA
operator sequ
ences. Previous biophysical analysis revealed a homo
-
dimeric protein that
binds to a pseudo
-
symmetrical sequence of DNA [1]. However each of the three binding sites
has a different symmetry based around a 2 or 3bp spacer between the “c
-
boxes”. The HTH
s
tructure of the protein allows for some flexibility in the loop between the recognition and
scaffold helices. Recent high resolution crystal studies of single operator sites has shown
that in the less symmetrical operator the loops are in different confor
mations in a single dimer
and a more symmetrical sequence leads to a more symmetrical loop configuration [2].



A mutagenic approach was used to investigate the role and importance of the individual
amino acids in and around the recognition helix. A comb
ination of high resolution
crystallography and DNA binding studies has allowed us to probe the underlying basis of
these protein
-
DNA interactions.


[1] J.E. McGeehan,
et al
.
(2008).
Nucleic Acids Res

36
, 4778
-
4787
1.

[2]

J.E. McGeehan,
et al
.
(2012)
.

Nucleic Acids Res
. In Press
.























SWSBC 2012 University of Southampton


36

14)
The Basis for Carbapenem Hydrolysis by Class A b
-
Lactamases: A Combined
Investigation using Crystallography and Simulations

Fátima Fonseca
1,2
, Ewa I. Chudyk
1
, Marc W. van der Kamp
1
, António Correia
2
,
Adrian J.
Mulholland
1

and
James Spencer
1


(1) University of Bristol

(2) University of Aveiro, Portugal


Carbapenems are the most potent b
-
lactam antibiotics and key drugs for treating infections by
Gram
-
negative bacteria. In such organisms, b
-
lactam
resistance arises principally from b
-
lactamase production. The class A b
-
lactamases, the most prevalent of these enzymes, are
generally inhibited by carbapenems as the covalent acylenzyme intermediate deacylates very
slowly. However, carbapenem
-
hydrolyzing

class A b
-
lactamases are now disseminating in
clinically relevant bacteria. The reasons why carbapenems are substrates for some, but inhibit
other, class A b
-
lactamases, remain to be fully established.

We have determined crystal structures of the class A
carbapenemase SFC
-
1 from
Serratia
fonticola

and of two mutants that trap the Michaelis and covalent acylenzyme complexes of
the carbapenem meropenem. The SFC
-
1 meropenem acylenzyme adopts an orientation
distinct from that observed in carbapenem
-
inhibited e
nzymes. Molecular dynamics
simulations indicate this mode of binding is favoured by the wild
-
type Michaelis and
acylenzyme complexes. Structural comparisons show that subtle repositioning of key
residues (Ser70, Ser130, Asn132 and Asn170) enlarges the SFC
-
1 active site to permit this
reorientation of the carbapenem 6a
-
1R
-
hydroxyethyl group, preventing its interaction with the
water molecule responsible for deacylation and promoting efficient carbapenem hydrolysis.








15)
Human Peroxiredoxin and
Oxidative Stress

Paul James
, Misha Isupov and Jennifer Littlechild


Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental
sciences, University of Exeter, Stocker Road, Exeter EX4 4QD


Peroxiredoxins (Prx) are a family of
multifunctional enzymes that protect against oxidative
stress and are produced in very high levels in cells. Prx form part of a peroxide scavenging
system along with Sulfiredoxin (Srx), Thioredoxin (Trx) and Thioredoxin reductase (TrxR). All
these enzymes
use redox active cysteine residues to detoxify reactive oxygen species or
reduce the other enzymes in the system.The structure of the red blood cell hPrxII was solved
in Exeter several years ago and showed a decameric structure with the peroxidatic cystein
e
residue oxidised to sulfinic acid.

A new structure has been solved that shows the decameric form of hPrxII in the disulfide state
that is known to form as part of its active cycle. This has revealed the necessary movements
that can occur to bring the per
oxidatic and resolving cysteine residues together (previously
~13Å apart) to form the disulfide bond. It was previously believed that the decameric form ofd
the protein could not exist in the disulfide state and only formed when the Prx was a dimer.

In a
ddition an attempt was made to form the potential complexes between hPrxII and Trx, and
hPrxII and Srx. This was successful and has provided new information on the stoichiometry of
binding but to date diffraction quality crystals have not been obtained.







SWSBC 2012 University of Southampton


37

16)
Purification and X
-
ray Diffraction Studies of the Oxygenase Component of
Pseudomonas putida
2,5
-
Diketocamphane 1,2
-
Monooxygenase in Complex with FMN

Vahid Saneei
, Michail N. Isupov, Stephlina Dcunha and Jenny A. Littlechild


University of Exeter


Camphor degradation in
Pseudomonas putida
depends on three different Baeyer
-
Villiger
monooxygenases (BVMO). The 2,5
-
diketocamphane 1,2
-
monooxygenase enzyme degrades
the 2,5
-
diketocamphane which is formed during degradation of (+)
-
camphor and a related 3,
6

diketocamphane 1,2
-
monooxygenase enzyme degrades the 3,6
-
diketocamphane formed
from (
-
) camphor. These enzymes belong to type II group of BVMOs which use the cofactors
FMN and NADH for activity. Both genes are located on a large CAM plasmid in
Pseudomon
as
putida.
Both enzymes are formed by a dimeric oxygenase component and a flavin reductase
component. The oxygenase components of each enzyme have a molecular weight of 7
8k
Da
and binds to one FMN cofactor molecule.

The oxygenase components of both enzymes
have been cloned and over
-
expressed. They
have been purified, crystallised, and their three dimensional structure in complex with FMN
determined using X
-
ray crystallography. The enzyme crystals belongs to P12
1
1 space group
with unit cell dimensions of a=51
.89, b=89.72, c=75.17, and α=90.00°, β=100.45°, γ=90.00°,
and diffracted to 1.79Å.

A NADH dependent FMN reductase, MsuE, has been cloned from the
Pseudomonas

genome
and the recombinant protein has been used to obtain a complex with the oxygenating
subunits
. A complex has been observed by gel filtration experiments and crystallisation
studies are underway
.








17)
Inhibition mechanism of human galectin
-
7 by a novel galactose
-
benzylphosphate
inhibitor.

Geoffrey Masuyer
, Talat Jabeen, Christopher T Oberg,
Hakon Leffler, Ulf J Nilsson, K Ravi
Acharya.


University of Bath


Galectins are involved in many cellular processes due to their ability to bind carbohydrates.
Understanding their functions has shown the necessity for potent and specific galectin
inhibitors. Human galectin
-
7 (hGal
-
7), in particular, has been highlighted as an important
marker in many types of cancer by either inhibiting or promoting tumour growth. Producing
ligands able to selectively target hGal
-
7 will offer promising tools for de
ciphering cancer
processes in which hGal
-
7 is involved as well as present potential solutions for future
therapeutics. Here we report the high resolution crystal structure of hGal
-
7 in complex with a
synthetic 2
-
O
-
benzylphosphate
-
galactoside inhibitor (whi
ch is > 60
-
fold more potent than its
parent galactoside). The high resolution crystallographic analysis highlights the validity of
using saccharide derivatives, conserving properties of the galactose binding, while enhanced
affinity and specificity is prov
ided by the added phosphate group. This structural information
will allow the design of further inhibitors with improved potency and specificity.








SWSBC 2012 University of Southampton


38

18)
Structural characterization of angiotensin I
-
converting enzyme in complex with a
selenium analogue
of captopril

Mohd Akif,
Geoffrey Masuyer
, Sylva L U Schwager, BhaskarJ Bhuyan, Govindasamy
Mugesh, R Elwyn Isaac, Edward D Sturrock, K Ravi Acharya.


University of Bath


Human somatic angiotensin I
-
converting enzyme (ACE), a zinc
-
dependent dipeptidyl

carbo
xypeptidase, is central to the regulation of the renin

angiotensin aldosterone system. It
is a well
-
known target for combating hypertension and related cardiovascular diseases. In a
recent study by Bhuyan and Mugesh [Org. Biomol. Chem. (2011) 9, 1356

1365]
, it was
shown that the selenium analogues of captopril (a well
-
known clinical inhibitor of ACE) not
only inhibit ACE, but also protect against peroxynitrite
-
mediated nitration of peptides and
proteins. Here, we report the crystal structures of human testi
s ACE (tACE) and a homologue
of ACE, known as AnCE, from Drosophila melanogaster in complex with the most promising
selenium analogue of captopril (SeCap) determined at 2.4 and 2.35 Å resolution, respectively.
The inhibitor binds at the active site of tACE

and AnCE in an analogous fashion to that
observed for captopril and provide the first examples of a protein

selenolate interaction.
These new structures of tACE

SeCap and AnCE

SeCap inhibitor complexes presented here
provide important information for furt
her exploration of zinc coordinating selenium
-
based ACE
inhibitor pharmacophores with significant antioxidant activity.






19)
Probing the Conformational Dynamics and Transfer Pathway of Ions and
Substrates Through the NorM MATE Transporter
via

Multiscale Molecular Dynamics
Simulations

Pete Yuk Ming Leung
1
, Daniel Holdbrook
1
, Thomas Piggot
1
, Oliver Beckstein
2

and Syma
Khalid
1


(1)

School of Chemistry, Highfield Campus, University of Southampton, Southampton SO17
1BJ, UK

(2)

Arizona State Univers
ity, Department of Physics, P.O. Box 871504, Tempe, AZ


The multidrug and toxic compound extrusion (MATE) transporters are the latest inclusion in
the
multidrug efflux transporter family. NorM, a MATE transporter from
Vibrio cholerea
,
consists of 461 amino

acid residues with 12 transmembrane helices and is a secondary
transporter; it uses the difference in the electrochemical potential of a Na
+

ion across the
membrane for drug extrusion [
1
]. However, the exact mechanism by which it achieves this is
still not known at the molecular level. Recent experimental studies have shown that the MATE
transporter plays a role in mult
idrug resistance in bacteria and mammals through extruding
drug out of the cell. Using the outward
-
facing X
-
ray structure of NorM [
2
] and the inverted
-
topology repeats method [
3
], we have generated a model of the NorM inward
-
facing
co
nformation, whose structure is not currently known. Molecular dynamics simulations have
been employed to probe the mechanisms of conformational change in NorM and the transfer
pathway of the ion and substrate (drug) into and out of the cell.


[
1
]

Kuroda, T
. and T. Tsuchiya,
Multidrug efflux transporters in the MATE family.

Biochimica
et Biophysica Acta (BBA)
-

Proteins & Proteomics, 2009.
1794
(5): p. 763
-
768.

[2]

Xiao He, P.S., Andrey Karyakin, Mariah Evin, Wen
-
Xu Hong, Qinghai Zhang, and Geoffrey
Chang,
Structure of a Cation
-
bound Multidrug and Toxic Compound Extrusion Transporter.

Nature, 2010.
467
(7318): p. 991
-
994.

[3]

Radestock, S. and L.R. Forrest,
The Alternating
-
Access Mechanism of MFS Transporters
Arises from Inverted
-
Topology Repeats.

Journal of
Molecular Biology, 2011.
407
(5): p. 698
-
715.




SWSBC 2012 University of Southampton


39

20)
A Structural Analysis of T
-
Cell escape by HIV

Anna Fuller
, David Cole, Pierre Rizkallah, Andrew Sewell.


Cardiff University


Human immunodeficiency virus (HIV) can infect and eliminate CD4+ T
-
cells.
Depletion of
these cells, that form an important wing of the adaptive cellular immune system, is a major
factor leading to acquired immune deficiency syndrome (AIDS). CD8+ T
-
cells have the ability
to eliminate cells infected with HIV and other viruses thro
ugh an interaction between the T
-
cell
receptor (TCR) and peptide
-
major histocompatiblity complex class

I (pMHCI) at the cell
surface.

However, HIV can rapidly mutate many of its protein components to avoid detection by
circulating CD8+ T
-
cells. The HLA A*0
201 restricted HIV
-
GAG epitope (SLYNTVATL) (A2
-
SLY) has been shown to be a major target for CD8+ T
-
cell immune responses. A number of
escape variants derived from this epitope have been extensively investigated. In order to
study the structural basis of HI
V immune escape based on this epitope, we have solved the
structure of the a TCR (868 TCR), derived from an A2
-
SLY specific CD8+ T
-
cell clone (868),
alone and in complex with wildtype A2
-
SLY, and 2 well characterized escape mutant variants
(A2
-
SLY
6I
; SLYNT
I
ALT and A2
-
SLY
3F6I8V
; SL
F
NT
I
A
V
T). These novel data provide
important insights into the alterations in the structural elements of the A2
-
SLY variants during
binding to the 868 TCR that lead to evasion of CD8+ Tcell mediated immunity by HIV.










21)
St
ructural plasticity and antigen selection by MHC class I

L. Bolton
,

S. Findlow

, T. Elliott, and J.M. Werner


Centre of Biological Sciences, University of Southampton, Southampton, SO17 1BJ


The selection of immunogenic peptides by MHC class I molecules in the endoplasmic
reticulum has major implications for immune system stimulation and may present an
intervention point to treat disease. The selection process is mediated by a weakly
-
interacti
ng
multi
-
protein peptide loading complex, the central role of which is the modulation of MHC
class I conformation.

Based on time
-
resolved cell biology experiments, computational models have been
developed to describe the kinetic control of MHC class I pep
tide selection (Dalchau et al,
2011,
A peptide filtering relation quantifies MHC class I peptide optimisation). These indicate
the presence of an intermediate in peptide binding.

We investigate the structural dynamics of the MHC
-
peptide complex using NMR, a technique
that is uniquely placed to detect structural plasticity that cannot be observed in X
-
ray crystal
structures. High
-
quality spectra of
2
H
13
C
15
N
-
labelled MHC class I have

enabled the
assignment of the h
β
2
M subunit, permitting the comparison of h
β
2
M structure in the free and
bound states.










SWSBC 2012 University of Southampton


40

22)
Computational and expression studies of the
Chlamydia pneumoniae

Major Outer
Membrane Protein (MOMP)

Francis O Atanu
, Ian
M Jones and Kimberly A Watson


School of Biological Sciences, University of Reading, UK


The Major Outer Membrane Protein (MOMP) of the genus
Chlamydia

is a cysteine rich 40
kDa protein localised to the outer membrane.
C. pneumoniae
is a human pathogen
res
ponsible for at least 20 % of lower respiratory tract infections. Its role in the development of
atherosclerosis has been justified by several lines of evidence. Recently, the anti
-
atherosclerotic activities and modulation of cell mediated immunity by reco
mbinant MOMP
from
C. pneumoniae

was demonstrated.

In this report, we present a three dimensional homology model of MOMP based on the fatty
acid transporter of
E. coli

and the most plausible knowledge based assignment of function
based on orientation of id
entified motifs. We tested the hypothesis of cell mediated
modulation of immunity by preferentially docking four MOMP derived peptides unto Major
Histocompatibility Complex (MHC) class II.

Our results were corroborated by predictions for immunoinformatic
applications. Expression
and purification studies of MOMP suggest the likelihood to localise to the inner membrane
when expressed in non
-
Chlamydial

host and purifiable at ~100 µg per liter.







23)
Membrane Protein Laboratory at Diamond
-

What we can do

for you

Isabel Moraes


Membrane Protein Laboratory
,
Diamond Light Source Ltd


Membrane proteins are the supreme example where more effort is needed in structural
biology. In spite of their abundance and importance, of the more than 60,000 protein
structures in the Protein Data Bank, only around 200 of these structures represent unique
membrane proteins.

To facilitate structural studies on membrane proteins, The Membrane Protein Laboratory at
Diamond Light Source Ltd (Diamond
-
MPL) was created in a c
ollaboration between Imperial
College London and Diamond, funded by the Wellcome Trust.

The MPL is a research and training state
-
of
-
the
-
art user facility open to scientists from
laboratories anywhere in the world interested in solving the 3
-
dimensional str
uctures of
membrane proteins by X
-
ray crystallography.
Because membrane proteins are unstable, hard
to crystallise and crystals difficult to handle. More systematic approaches and technical
developments are needed to improve the success rate of the structu
re determination of
membrane proteins.

The MPL has a formal collaboration with I24 microfocus beamline at Diamond to develop new
techniques for crystallisation and structural determination of membrane proteins, including
prototype systems for high throughp
ut methods, improving handling of small and delicate
crystals, and methods for collecting and merging data from a large number of small crystals.

The beamline is unique in its ability to deliver a tunable X
-
ray beam (6


25keV) of variable
size between 5 µ
m and 50 µm on to crystal samples. This versatility is coupled with a state
-
of
-
the
-
art pixel array detector.







SWSBC 2012 University of Southampton


41

24)
Preliminary crystal diffraction of the
Vibrio cholerae

Hfq protein

Jack Phillips
, Anastasia Callaghan, John McGeehan.


University of
Portsmouth


Hfq protein from Vibrio cholerae is a novel antibacterial target. Cholera disease is caused by
the pathogenic bacterium Vibrio cholerae by colonising the intestines and releasing the
cholera toxin which causes diarrhoea. V. cholerae modulates its behaviour

from colonisation
to pathogenesis by post
-
transcriptional gene regulation. This regulation is mediated by Hfq,
which can be found in many other pathogenic bacteria and so Hfq presents itself as an
important target that can be utilised in combating cholera

disease as well as other enteric
disease
-
causing pathogens.

Hfq is an ideal antibacterial target because there is potential to attack a bacteria’s ability to
become virulent without introducing an evolutionary selective pressure for an antibiotic
-
resista
nt strain because the bacteria would not be killed by the drug.

We have so far purified the protein from over
-
expressing in E. coli and have crystallised the
protein. The protein crystals diffract but the diffraction is highly anisotropic and needs
optimis
ing.

Experiments to improve the diffraction are currently being carried out as well as further
screening for optimum crystallisation conditions.



25) Malarial vitamin B6 biosynthesis

Ivo Tews
1,2
, Gabriela Guédez
2
, Volker Windeisen
2
, Irmgard Sinning
2
,
Katharina Hipp
3
, Bettina
Böttcher
3
, Martin Gengenbacher
4
, Bianca Derrer
5
, Barbara Kappes
5


(1) University of Southampton, Centre for Biological Sciences, Southampton SO17 1BJ

(2) Heidelberg University Biochemistry Center, INF328, 69120 Heidelberg, Germany

(3)
University of Edinburgh, School of Biological Sciences, Mayfield Road, Edinburgh EH9
3JR

(4)
Max
-
Planck
-
Institute for Infection Biology, Charitéplatz 1, 10117 Berlin, Germany

(5)
University Erlangen
-
Nürnberg, Paul
-
Gordan
-
Str. 3, 91052 Erlangen, Germany



It is perhaps surprising that the main route of vitamin B6 biosynthesis is a recent discovery [1].
The present biochemical research addresses the capability of the enzyme PLP synthase to
synthesize vitamin B6 directly from two carbohydrates and the amin
o acid glutamine.
Understanding this enzyme is of relevance in anti
-
microbial and anti
-
parasitic drug discovery,
particularly in the search for new strategies in anti
-
malarial therapy. Our initial crystallisation
attempts with malarial enzymes did not yiel
d crystals suitable for 3D structure determination.
When we studied the assembly of the 690 kDa protein complex by electron microscopy we
realised that the PLP synthase complexes had aggregated into fibres, which hindered
crystallisation [2]. These problem
s were obviated with the study of a chimeric complex made
up of
Plasmodium falciparum
and
Plasmodium berghei

proteins that was enzymatically viable.
Compared with previously determined bacterial structures [3, 4] the
Plasmodium

structure
showed a different

positioning of enzymatic subunits relative to each other, explaining
differences in subunit association noted earlier using isothermal calorimetry. These
differences may be exploitable in drug design. A substrate complex at 2.4 Å resolution shows
carbohyd
rate attachment at the pentose C1 atom through Schiff base formation. While we
know that PLP synthase has a cooperative hexameric core, our EM data now suggest that
association of the glutaminase subunit with the core complex is at random.


[1] Fitzpatrick
, T. B., Amrhein, N., Kappes, B., Macheroux, P., Tews, I. & Raschle, T. (2007).

The Biochemical journal

407
, 1
-
13.

[2] Guedez, G., Hipp, K., Windeisen, V., Derrer, B., Gengenbacher, M., Bottcher, B., Sinning,
I., Kappes, B. & Tews, I. (2012).

Structure

20
, 172
-
184.

[3] Strohmeier, M., Raschle, T., Mazurkiewicz, J., Rippe, K., Sinning, I., Fitzpatrick, T. B. &
Tews, I. (2006).

Proc Natl Acad Sci U S A

103
, 19284
-
19289.

[4] Zein, F., Zhang, Y., Kang, Y. N., Burns, K., Begley, T. P. & Ealick, S. E. (2006).

Biochemistry

45
, 14609
-
14620.

SWSBC 2012 University of Southampton


42

26) The relevance of cyanobacterial Tic22 to plant and malarial protein transport.

Ivo Tews
1,2
, Patrick Koenig
2
, Irmgard Sinning
2
, Johanna Tripp
3
, Oliver Mirus
3
, Enrico Schleiff
3



(1) University of Southampton, Centre for

Biological Sciences, Southampton SO17 1BJ

(2) Heidelberg University Biochemistry Center, INF328, 69120 Heidelberg, Germany

(3)
Goethe University Frankfurt, Department of Biosciences, 60438 Frankfurt, Germany


Prokaryotic and eukaryotic cell membranes cont
ain specific transporters for proteins to
mediate transfer across and insertion into bio
-
membranes [1]. One can compare the
transporters found in organelles such as mitochondria or plastids with the ones found in
bacteria. For instance, membrane
-
spanning b
eta
-
barrel pore subunits are present in
mitochondria (SAM50 and Tom40 [2]), in chloroplasts (Toc75 [3]), and in bacteria (Omp85 [4]).
An interesting problem exists with respect to the directionality of transport, which must have
changed during evolution if

all these proteins should have common ancestry. We had
previously addressed this with the study of the so
-
called POTRA domains (POlypeptide
-
TRansport
-
Associated) of a cyanobacterial Omp85 protein [5], and have now extended these
studies to the cyanobacter
ial protein Tic22. The crystallographic 3D structure has a “butterfly”
shape revealing a repeat likely caused by gene duplication [6]. Four helices point orthogonal
at each other, adding up their dipole moments in a central cavity. The surface of the struc
ture
is dotted with hydrophobic pockets in which we identified bound solvent molecules. While the
functional significance of these features is not yet clear, it is likely that they represent binding
sites for protein substrates, and Tic22 is thus assigned
a chaperone function. We
demonstrate that Tic22 is present in the cyanobaterial periplasm as well as in thylakoids, and
it can be functionally replaced by knock
-
in of a plant orthologue. The structural clues together
with the functional data suggest that T
ic22 can have a function in both, protein import or
protein insertion, depending on the organism where it is found. The protein is conserved in
bacteria, plants, and unicellular organisms such as the plastid containing apicomplexa with
the important human
pathogen
Plasmodium
, the causative agent of malaria. It therefore links
back these protein transporters to a common ancestry.


[1]
Bohnsack, M. T. & Schleiff, E. (2010).

Biochimica et biophysica acta

1803
, 1115
-
1130
.

[2]

Chacinska, A., Koehler, C. M., Milenkovic, D., Lithgow, T. & Pfanner, N. (2009).

Cell

138
,
628
-
644
.

[3]
Oreb, M., Tews, I. & Schleiff, E. (2008).

Trends in cell biology

18
, 19
-
27.

[
4]
Knowles, T. J., Scott
-
Tucker, A., Overduin, M. & Henderson, I. R. (2009
).

Nature reviews.
Microbiology

7
, 206
-
214.

[5] Koenig, P., Mirus, O., Haarmann, R., Sommer, M. S., Sinning, I., Schleiff, E. & Tews, I.
(2010).

The Journal of biological chemistry

285
, 18016
-
18024.

[6]
Tripp, J., Hahn, A., Koenig, P., Flinner, N., Bublak, D., Brouwer, E.M., Ertel, F., Mirus, O.,
Sinning, I., Tews, I. & Schleiff, E. (2012)
The Journal of biological chemistry
, in press.















SWSBC 2012 University of Southampton


43

27)
Thermophilic Enzymes and Applications in Commercial
Biocatalysis

Jenny Littlechild


The Henry Wellcome Building for Biocatalysis, University of Exeter, Stocker Road, Exeter,

EX4 4QD, UK.


Naturally thermostable proteins are finding increasing applications in commercial biocatalysis.
In the Biocatalysis Cent
re in Exeter we have worked on several enzymes over the years;
some of which are already used for the production of pharmaceutical intermediates. These
enzymes form part of ‘white biotechnology’


a terminology used by the EU. Cloning, over
-
expression, bio
chemical and structural analysis of enzymes of interest can reveal factors
important for thermostability and substrate specificity.

When non
-
natural substrates are used different classes of enzyme can carry out the catalytic
turnover. This is the case for

gamma lactam cleavage which can be carried out by a signature
amidase from the thermophilic archaeon,
Sulfolobus

species and a novel zinc enzyme related
to formamidase from the mesophile,
Comomonas

species. Both enzymes use different
mechanisms and have d
ifferent structures. At non
-
physiological pH, side reactions can be
carried out which are different from the activity at pH 7.0. Another gamma lactamase from
Aureobacterium
species with opposite stereoselectivity is a αβ hydrolase fold esterase but
can car
ry out a bromination reaction at pH 5.0.

Exeter is part of a large consortium grant called ‘Hotzyme’ where both metagenomics and
new novel thermophilic genomes are being used to screen for new thermostable enzymes
which can be identified from both bioinfor
matic and high throughput screening approaches.









28)
Cryop
rotection screening made simple

Enrico Stura
1
, Laura Vera
1

and Paul Thaw
2


(1)
Institut de Biologie et des Technologies, iBiTec
-
S ; 91191 Gif /Yvette Cedex, France.

(
2)

Molecular Dimensions, Unit 6, Goodwin Business Park, Newmarket, Suffolk, UK.


Finding a cryoprotecting solution that “works” is not as difficult as finding one that “really
works”. Several post crystallization manipulations can increase the quality of th
e data that can
be collected in house or at synchrotron sources: dehydration, annealing and the use of a
good cryoprotecting solution. Typically it is too laborious to screen through the multitude of
various alternative possibilities for each crystal form
and it is common to accept the first
cryoprotecting solution that “works”.

However, if the selection of the optimal cryoprotectant solution is done over several
macromolecular crystals, it becomes possible to evolve cryoprotecting solutions that give
exce
llent results over a large range of crystals, often without failure even on new project. This
iterative strategy aims to evolve the cryoprotectant mix using the “champion competition
method”. The most successful cryoprotectant mixes from previous data coll
ection are retained
and used in the following data collection sessions and competed against new precipitant
-
cryoprotectant
-
buffer combinations. Quickly “champion” solutions emerge but “challengers”
introduced at each data collection cycle get to compete to

become the new “champions”.








SWSBC 2012 University of Southampton


44

29)
Characterisation of Dehalogenases from a Marine Bacterium

Novak, H.
1
, Gotz, D.
2
, Mearns
-
Spragg, A
2
,Isupov, M.

1
and Littlechild, J. A.
1


(
1
)

The Henry Wellcome Building for Biocatalysis, University of Exeter, U.K.

(
2
)

Aquapharm Biodiscovery Ltd, European Centre for Marine Biotechnology, Dunstaffnage,
Oban, Argyll, PA37 1QA, U.K.


Many herbicides, fungicides and insecticides are halogenated. These chemicals are often
recalcitrant and therefore may pose a threat to the environment with additional concerns
including bioaccumulation. This makes dehalogenases an attractive class of enzy
mes that
can be used for bioremediation purposes.
Specifically, haloacid
dehalogenases are also of
great interest because they can produce optically pure 2
-
hydroxyalkonoic acids.


A marine bacterium from the proprietary collection of marine microorganisms

at Aquapharm
Biodiscovery Ltd tested positive for L
-
haloacid dehalogenase activity.
The genome has been
sequenced and an L
-
haloacid dehalogenase was identified. The protein has been cloned and
overexpressed in
E.coli
. The L
-
haloacid dehalogenase has been purified using nickel affinity
and gel filtration chromatography. Microbatch crystallization screens were conducted and
crystals formed in many conditions. The crystal structure was elucidated at 1.8 Å and the
enzyme
biochemically characterised to determine the substrate specificity, temperature
optimum, thermal stability and solvent stability. Biochemical and structural characterisation of
these enzymes will allow us to assess their industrial importance.







30) S
tructural and biochemical characterisation of MorA, involved in the regulation of
c
-
di
-
GMP levels in biofilm formation

C. W. Phippen
, J. Webb, B. Keevil, I. Tews.


University Of Southampton, Centre for Biological Sciences, Institute for Life Sciences
(IfL
S),
Highfield Campus, Southampton SO17 1BJ


Recent discoveries show that the signalling molecule cyclic diguanylate monophosphate (c
-
di
-
GMP) is involved in bacterial differentiation and in the regulation of biofilm formation. Within
biofilms, bacteria beco
me more tolerant towards antibiotics, leading to an enhanced evasion
of the host immune response, even under antibiotic treatment. Biofilm formation, therefore,
underlies many persistent human infections.

High levels of c
-
di
-
GMP switch bacteria from a plan
ktonic to a biofilm state. Bacteria often
contain several proteins that can synthesize c
-
di
-
GMP or hydrolyse c
-
di
-
GMP. Amazingly,
both activities are often found in the same protein. We study one such protein from
Pseudomonas aeruginosa
, an
opportunistic
human pathogen responsible for many biofilm
-
associated diseases, including chronic respiratory infection in cystic fibrosis patients.

The
MorA protein contains a four sensory PAS/PAC domains and is membrane anchored [1].

Here, we present initial results f
rom our structural work. The aim of the work is to characterise
the multifunctional MorA protein, and in particular understand how the PAS domains regulate
enzyme function. A sequence analysis of the PAS domains allows assigning their function.
Preliminary

results from our crystallisation experiments with various MorA constructs will be
shown.

[1] Choy WK, Zhou L, Syn CK, Zhang LH, Swarup S. (2004) J. Bacteriol. 186, 7221
-
7228.








SWSBC 2012 University of Southampton


45

31)
Structural basis of eEF2K activation by Calcium/Calmodulin

H.Mikolajek
1

and Kelly Hopper
1
, S. Rosset
1
, S. Knapp
2
, C. G. Proud
1

and J.M.Werner
1

(1)
Centre of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK

(2)
Structural Biology Consortium, Oxford OX3 7DQ, UK


Eukaryotic elongation factor
2 kinase (eEF2K) is a calcium/calmodulin (Ca
2+
/CaM)
-
dependent
protein kinase that phosphorylates and inactivates the translation elongation factor eEF2,
thereby slowing down the rate of translation elongation.

The protein consists of an unstructured N
-
te
rminal region containing the Ca
2+
/CaM binding
site, a highly atypical α
-
kinase domain, an unstructured central region with regulatory
phosphorylation sites and a structured C
-
terminal domain containing four Sel1
-
like repeats.
The structure and molecular me
chanism of regulation of this potential cancer target are not
known.

We have taken a divide and conquer approach to study the structure and regulation of this
enzyme using a high throughput (HT) parallel cloning and recombinant expression approach
for co
nstruct screening in conjunction with NMR spectroscopy.

The binding of Ca
2+
/CaM to different eEF2K constructs was investigated using NMR
spectroscopy to analyse the high affinity interaction between eEF2K and Ca
2+
/CaM. Taken
with recent biochemical finding
s
1

the data we present here demonstrate a first insight into the
molecular mechanism of regulation of eEF2K by CaM and Ca
2+
.

[1]

Pigott
et al
.

(2012)
Insights into the regulation of eukaryotic elongation factor 2 kinase and
th
e interplay between its
domains.

Biochem J
.

442
,
105
-
18






32)
The structure and substrate specificity of a thermophilic archaeal serine : pyruvate
aminotransferase from
Sulfolobus solfataricus

Chris Sayer
1
, Martin Bommer
2
, Michail N. Isupov
1
, John Ward
2

and Jennifer Littlechild
1


(1)
Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental
Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK

(2)
Research Department of

Structural and Molecular Biology, University College London,
Darwin

Building, Gower Street, London WC1E 6BT, UK


A putative aminotransferase from the thermophilic archaeon
Sulfolobus solfataricus

has been
cloned and over expressed in
Escherichia coli.
The recombinant enzyme has been
biochemically characterised and substrate specificity studies have confirmed its function as a
serine:puruvate aminotransferase.

The structure of the native form of the enzyme has been solved using the molecular
replacement

method
to 1.8
Å
resolution. The protein is a homodimer adopting the type
-
I fold
of pyridoxal 5'
-
phosphate (PLP)
-
dependent aminotransferases. The PLP co
-
factor is
covalently bound in the active site of the enzyme in the internal aldimine form. The enzyme
s
hows close structural resemblance to alanine:glyoxylate aminotransferases (EC 2.6.1.44).
The structure of the
S. solfataricus

enzyme was also determined in the presence of an
aminotransferase inhibitor, gabaculine. A comparison of the structure of
S. solfa
taricus

enzyme with alanine:glyoxylate aminotransferase has identified structural features which
could be responsible for differences in substrate specificity between the two enzymes.

The thermostability of the enzyme showed that after incubation at 80°C for 10minutes more
than 90% of activity is retained between pH 5.0 and 6.0. The structural studies have revealed
ion pair networks and hydrophobic interactions that could contribute to

the enzymes
thermostability.





SWSBC 2012 University of Southampton


46

33)
Origin of IGF2R/IGF2 binding: Using structure and dynamics to follow and enhance
evolution

Madeleine Strickland
1
, C. Williams
1
, R.Z. Ellis
1
, D. Rezgui
2
, H. Hoppe
2
, A.B. Hassan
2
, M.P.
Crump
1


(1) University of Bristol

(2) University of Oxford


Insulin
-
like growth factor 2 (IGF2) is a growth hormone responsible for cell proliferation mainly
during gestation, acting through the insulin receptor A and IGF1R. IGF2 is transported to the
lysosome for degradation by domain
11 of IGF2R, which controls its bioavailability.
Overexpression of IGF2 and mutation of domain 11 are both causes of cancer, providing
scope for a higher affinity domain 11 to be used as an anti
-
cancer therapeutic.

In order to determine how to improve the

interaction between IGF2 and domain 11 a
combination of evolutionary structure
-
function and dynamics studies have been carried out.
The origin of IGF2 binding occurred with the development of lactation and placentation in
early mammals, of which initial,

weak binding was first identified in the echidna. Later
mammals have a much stronger interaction with IGF2 due largely to the placement of
hydrophobic residues in the binding site and flexibility (or lack thereof) in the four binding
loops.

A library of

mutants was produced based on this knowledge, which were screened for high
affinity binding to IGF2 and a potential drug candidate was found that increased binding
almost 100 fold.






34)
Analysis of activator and substrate binding to eukaryotic
elongation factor 2 kinase
(eEF2K)

K. Hooper
, H. Mikolajek, G. Baker, S. Rosset, C.G. Proud and J.M. Werner


Centre for Biological Sciences, Institute for Life Sciences, University of Southampton,
Southampton, SO17 1BJ


Eukaryotic elongation factor 2 kinas
e (eEF2K) critically regulates translation elongation by
controlling the activity of eEF2, which catalyses the translocation reaction of the ribosome.
However, when eEF2 is phosphorylated its binding to the ribosome is inhibited and therefore
translation
elongation is blocked. eEF2K phosphorylates eEF2 and therefore prevents the
progression of translation elongation.

eEF2K is activated by elevated Ca
2+
levels via calmodulin (CaM). The molecular mechanism
of activation is not understood, however a CaM bin
ding motif at the N
-
terminus of eEF2K is
predicted as a key interaction site for CaM and therefore is important for the regulation of the
kinase activity.

We have investigated the interaction between CaM and the CaM binding motif of eEF2K
using NMR titrat
ions and isothermal titration calorimetry (ITC). The NMR titration data in
saturating Ca
2+
conditions suggests the presence of multiple peptide bound conformations
that may have implications for the activation process of eEF2K.

Aside from eEF2 the only kn
own substrate of eEF2K is a 16 residue peptide, MH
-
1, from
Dictyostelium.
We have determined the nature of the interaction between eEF2K and MH
-
1
using saturation transfer difference (STD) spectroscopy. The chemical shift assignments of
the MH
-
1 peptide identified critical chemical groups involved in the interaction with eEF2K.







SWSBC 2012 University of Southampton


47

35)
Optimisation of galacto
-
oligosaccharides through β
-
galactosidase engineering

Katerina Lazidou, Dimitris Charalampopoulos and Kimberly A Watson


University of Reading
,

Department of Food and Nutritional Sciences, School of Biological
Sciences


Prebiotic oligosaccharides are compounds with potential health benefits, and hence have
various commercial applications as nutraceutical supplements. Galacto
-
oligosaccharides
(GOS) are well established prebiotics, and are synthesised during lactose hydroly
sis by β
-
galactosidase, a reaction known as trans
-
galactosylation.

The research aim is to optimise transgalactosylation activity, hence GOS production, of two β
-
galactosidases: BbgIII (1935aa) and BbgIV (1052aa), which are produced by the industrial
strai
n
B.bifidum
NCIMB4117.

Sequence analysis was performed and the tertiary structure models of BbgIII and BbgIV were
determined. The models were structurally aligned to the
E. coli
template to determine the
active site residues. Results have revealed consistency between the conserved domain
organisation and the tertiary structure models. The active site predictions were promising.

Following the alignment, N
-
terminal and C
-
terminal t
runcations of BbgIII and BbgIV were
designed. Cloning, expression and purification of the constructs were conducted in a high
throughput way. Expression and purification of BbgIII full length enzyme and domains showed
higher expression levels of the constr
ucts, possibly correlating to a higher enzymatic activity.
However, for BbgIV constructs, low expression levels were observed, revealing a possibly
deleterious effect.







36)
Unpicking the molecular function of sedoheptulose
-
7
-
phosphate

Nicholas J
.

Harmer


Th
e Henry Wellcome Building for Biocatalysis, Stocker Road, Exeter EX4 4QD


Heptoses are found in the surface polysaccharides of most bacteria, contributing to structures
that are essential for virulence and antibiotic resistance. The biosynthetic
enzymes for these
sugars are therefore attractive targets for novel antibiotics. The best characterised
biosynthetic enzyme is sedoheptulose
-
7
-
phosphate isomerase (GmhA). This catalyses the
interconversion of sedoheptulose
-
7
-
phosphate and
D
-
glycero
-
D
-
manno
-
heptopyranose
-
7
-
phosphate, the first step in the biosynthesis of heptose. Structures of GmhA have been
solved from multiple organisms, and support an enzymatic mechanism involving residues
from three protomers of the GmhA tetramer in each active site. To
further understand the
mechanism of the enzyme, mutants of proposed active site residues were crystallised in the
presence of substrate. The crystal structures of five mutants highlighted the probable cause of
each mutant showing reduced activity. In parti
cular, one mutant shows the substrate poised to
react, helping to verify proposed mechanisms. Surprisingly, the D61A mutant, located in a
channel linking two active sites, shows only a subtle re
-
arrangement of water molecules;
however, this results in a ra
te reduction to 7% of original activity. To further understand this,
tetramers with four, three and one competent active site were prepared: these showed 100%,
100% and 50% activity respectively, suggesting that the GmhA tetramer acts as pairs of
mutually
inhibitory active sites.

[1] N. J. Harmer J. Mol. Biol.
400
, 379
-
92 (2010)







SWSBC 2012 University of Southampton


48


(37)
Probing

the

role

of

WcbI

protein

in

the

biosynthesis

of

the

Burkholderia

pseudomallei

capsular

polysaccharide.

Mirella Vivoli, Emily Ayres and Nicholas Harmer


School
of Bio
sciences, University of Exeter
, Stocker Road, Exeter EX4 4QD, UK.


Burkholderia

pseudomallei

is

a

facultative

intracellular

bacterial

pathogen

that

causes

melioidosis,

an

infectious

disease

that

is

endemic

and

often

fatal

in

several

tropical

and

subtropical

areas

such

as

Northern

Australia

and

South
-
East

Asia.

Moreover,

this

agent

is

considered

to

be

a

potential

bioterror

threat

and

is

listed

as

a

category

B

agent

by

the

US

Centers

for

Disease

Control

and

Prevention.

One

of

the

best

validated

virulence

factors

in

B.

pseudomallei

is

the

capsular

polysaccharide

(CPS),

an

extracelluar

polysaccharide

that

is

highly

attractive

for

vaccine

development.

The

24

genes

required

for

the

biosynthesis

of

CPS

have

been

identified.

One

of

them,

wcbI
,

produces

a

protein

with

no

homologues

of

known

function,

but

has

been

shown

to

be

required

for

the

virulence.

We

hypothesized

that

WcbI

acts

as

an

acetyltransferase,

an

activity

required

to

form

the

mature

capsule.

To

test

the

putative

role

of

this

protein

we

performed

both

enzymatic

and

structural

studies.

We

purified

the

protein

and

obtained

preliminary

crystals

that

diffract

strongly.

Moreover,

biochemical

data,

using

a

colorimetric

assay

that

detects

of

CoASH

generated

during

acetyl

transfer

by

reaction

with

5,5'
-
dithiobis(2
-
nitrobenzoate)

(DTNB),

revealed

that

WcbI

possesses

acetyltransferase

activity.

We

intend

to

resolve

the

crystal

structure

of

WcbI

and

test

its

activity

with

a

range

of

potential

substrates

to

give

fuller

insight

into

the

function

and

specificity

of

this

protein.

SWSBC 2012 University of Southampton


49

Delegate List


Name



Institution




Contact Email Address

Eyram Adjogatse

University College London


rmhaead@ucl.ac.uk

Chris Agnew


University of Bristol



c.agnew@bristol.ac.uk

Frank Atanu


University of Reading



f.o.atanu@pgr.reading.ac.uk

Jakrada Attarataya

University of Bristol



j.attarataya@bris.ac.uk

Stefan Bagby


University of Bath



bsssb@bath.ac.uk

Samuel Barker


University of Exeter



spb204@exeter.ac.uk

Maïwenn Beaugrand

Université du Québec à Montréa
l
missymaye@hotmail.com

Mark Benson


Rigaku Europe




mark.benson@rigaku.com

Nils Berglund


University of Southampton


nb9g08@soton.ac.uk

Richard Birkinshaw

University of Bristol



r.birkinshaw@bristol.ac.uk

Louise Bolton


University of Southampton


llb2v07@soton.ac.uk

William Bradshaw

University of Bath



wb268@bath.ac.uk

Leo Brady


University of Bristol



l.brady@bristol.ac.uk

Nick Burton


University of Bristol



nick.burton@bristol.ac.uk

Louise Butt


University of Portsmouth


louise.butt@port.a
c.uk

Matthew Byrne


University of Bristol



matt.byrne@bristol.ac.uk

Anastasia Callaghan

University of Portsmouth


anastasia.callaghan@port.ac.uk

Martin Challand


University of Bristol



Martin.Challand@bristol.ac.uk

Christopher Chambers

University of B
ath



cjc46@bath.ac.uk

Liz Clark


University of Bath



e.a.clark@bath.ac.uk

Alun Coker


University College London


alun.coker@ucl.ac.uk

David Cole


Cardiff University



coledk@cf.ac.uk

Peter Collins


Marresearch GmbH



peter@marresearch.com

Jon Cooper


UCL





jon.cooper@ucl.ac.uk

Susan Crennell


University of Bath



bsssjc@bath.ac.uk

Matthew Crump


University of Bristol



matt.crump@bristol.ac.uk

Ken Cunningham

Avid Nano




ken.cunningham@avidnano.com

Rosamund Ellis


University of Bristol



re5854@bristol.ac.uk

Sarah Embleton


University of Bath



se319@bath.ac.uk

Giannina Espina

University of Bath



gaes20@bath.ac.uk

Jon Extance


University of Bath



je208@bath.ac.uk

Stuart Findlow


University of Southampton


isf@soton.ac.uk

Anna Fuller


Cardiff University



whitea4@cardiff.ac.uk

David Hall


Diamond light source



david.hall@diamond.ac.uk

Nicholas Harmer

University of Exeter



N.J.Harmer@exeter.ac.uk

Charlotte Harrison

University of Bath



ch571@bath.ac.uk

Rob Hill



Bruker UK




rob
.hill@bruker.co.uk

Chris Hills


Uni of Bath




cah22@bath.ac.uk

Christopher Holland

Cardiff University



hollandcj@cardiff.ac.uk

Kelly Hooper


University of Southampton


kjh1g08@soton.ac.uk

Leyla Hussein


University of Bristol



leyla.hussein@bristol.ac.uk

Misha Isupov


University of Exeter



misupov@exeter.ac.uk

Paul James


University of Exeter



p.b.c.james@exeter.ac.uk

Geoff Kneale


University of Portsmouth


geoff.kneale@port.ac.uk

Katerina Lazidou

University of Reading



k
aterina.lazidou@gmail.com

Stephanie Liang

University of Bath



stl23@bath.ac.uk

Jennifer Littlechild

University of Exeter



J.A.Littlechild@exeter.ac.uk

Nia Marrott


University of Bath



nlh20@bath.ac.uk

Richard Martin


University of Portsmouth


richar
d.martin@port.ac.uk

Geoffrey Masuyer

University of Bath



gm283@bath.ac.uk

SWSBC 2012 University of Southampton


50

Name



Institution



Contact Email Address

John McGeehan

University of Portsmouth


john.mcgeehan@port.ac.uk

Wayne McQuaig

Taylor and Francis



wayne.mcquaig@tandf.co.uk

Halina Mi
kolajek

University of Southampton


hm1a08@soton.ac.uk

John Miles


Cardiff University



milesjj@cardiff.ac.uk

Charlotte Millership

Uni
versity of London, Queen Mary's
c.millership@qmul.ac.uk

Isabel Moraes


Imperial College/



isabel.de
-
moraes@diamond.ac.uk

Diamond Light Source


Marta Morais


University of Bath



mppm20@bath.ac.uk

Manjeet Mukherjee

National University of Singapore



manjeet.mukherjee@nus.edu.sg

Halina Novak


University of Exeter



h.r.novak@ex.ac.uk

Donald Ogg


Alpha Biotech




donald@alphabiotech.co.uk

Tram Pham


University of Bath



ttkp20@bath.ac.uk

Jack Phillips


University of Portsmouth


jack.phillips@port.ac.uk

Curtis Phippen


Southampton University



cwp2g08@soton.ac.uk

Andy Pickford


University of
Portsmouth


andy.pickford@port.ac.uk

Paul Race


University of Bristol



paul.race@bristol.ac.uk

Sneha Ramaswamy

University of Bath



sr511@bath.ac.uk

Pierre Rizkallah


Ca
rdiff University Medical School
rizkallahp@cardiff.ac.uk

Alice Robson


University
of Bristol



a.robson@bristol.ac.uk

Ramya Salimraj


University of Bristol



rs8667@bristol.ac.uk

Vahid Saneei


University of Exeter



vahid.saneei@gmail.com

Christopher Sayer

University of Exeter



cs402@ex.ac.uk

Andrea Schauenburg

Cardiff University



a.schauenburg@gmail.com

Mikhail Shevtsov

University of Portsmouth


misha.shevtsov@port.ac.uk

Peter Schoolingin
-
Jordan University of Southampton


pmsj@soton.ac.uk

Sunny Singh


University of Sussex



sunnykumarsingh@gmail.com

Dimitry Slyusarenko

Leica
Microsystems


dimitry.slyusarenko@leica
-
microsystems.com

James Spencer


University of Bristol



Jim.Spencer@bristol.ac.uk

George Stephens

Alpha Biotech




george@alphabiotech.co.uk

Madeleine Strickland

University of Bristol



m.strickland@bristol.ac
.uk

Ania Swiderska


University of Portsmouth


anna.swiderska@port.ac.uk

Sharon Tay


University of Bath



pst29@bath.ac.uk

Garrick Taylor


University of Oxford



garrick.taylor@bioch.ox.ac.uk

Ivo Tews


University of Southampton


I
vo.tews@soton.ac.uk

Paul Thaw


Molecular Dimensions



paul@moleculardimensions.com

Marisa Till


University of Bristol



marisa.till@bristol.ac.uk

Phillipa Timmins

Leica Microsystems


phillipa.timmins@leica
-
microsystems.com

Jean van den Elsen

University of Bath



bssjmh
ve@bath.ac.uk

Mirella Vivoli


University of Exeter



M.Vivoli@exeter.ac.uk

Esther von Schulthess

University of Southampton


elvs1g10@soton.ac.uk

Kim Watson


University of Reading



k.a.watson@reading.ac.uk

Joern Werner


University of Southampton


jmwe@soton.ac.uk

Aaron Westlake


University of Exeter



polosonastraw@yahoo.co.uk

Philip Williamson

University of Southampton


P.T.Williamson@soton.ac.uk

Marcus Winter


Agilent Technologies



marcus.winter@agilent.com

Steve Wood


UCL





s.wood@ucl.ac.
uk

Yi Yang








lanceyangyi@yahoo.com.cn

Pete Yuk Ming Leung

University of Southampton


yml1v07@soton.ac.uk