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2 Οκτ 2013 (πριν από 3 χρόνια και 8 μήνες)

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Exploring the Biology of Disulfide
-
Rich
Hyperthermophiles through Protein
Phylogenetic Profiles

Navapoln Ramakul
1
, Morgan Beeby
12
, and Todd O. Yeates
123

Southern California Bioinformatics Summer Institute

1
Department of Chemistry and Biochemistry,
2
Department of Energy Center for Genomics and Proteomics, and
3
Molecular Biology Institute, University of California, Los Angeles, CA 90095
-
1569

UCLA Bioinformatics:

Yeates Lab


Goals
: determine and analyze the three
-
dimensional structures of proteins.



Research
: focus on protein structure & function,
protein sequence & evolution, and protein
assembly & design.



Methods
: crystal structure determination
through theoretical and computational methods.

General Overview


Genomic Databases
: create opportunities for
new kinds of computational analyses and novel
discoveries.



Advantage
: special comparative studies using
multiple genomes to compare sequence vs.
structure.



Present Research
: investigate the surprising
revelation about disulfide bonds in certain
microbes from comparative studies.

Protein Disulfide Bonds


Previously believed to be prominent only
outside the cell.


Inside the cell


Disulfides only rarely found.


Disulfides are transient or functionally
important, rather than stabilizing.


Outside the cell


Abundant.


Intro



Comput.
Methods



Results &
Significance



Applications
& Future
Directions



Summary

Recent Studies


Unexpected disulfides in an intracellular protein.


Crystal structure of adenylosuccinate lyase
(ASL) from the
P. aerophilum

surprisingly
shown a protein chain stabilized by three
disulfide bonds (Toth
et al.,

JMB (2000) 301,
433
-
450.).

Toth
et al.,

JMB (2000) 301, 433
-
450

Disulfide bond

Disulfide bond


Intro



Comput.
Methods



Results &
Significance



Applications
& Future
Directions



Summary

Evidence for Abundant S
-
S bonds
in
P. aerophilum

Mallick, Boutz, Eisenberg, and Yeates (2002). PNAS 99, 9679
-
9684

Proteins with Even # of Cysteines


Intro



Comput.
Methods



Results &
Significance



Applications
& Future
Directions



Summary

Disulfide Abundance in Various
Genomes

Genome f (S
-
S)

Pyrobaculum aerophilum 0.44

Aeropyrum pernix 0.40

Pyrococcus abyssi 0.31

Pyrococcus horikoshii 0.28

Aquifex aeolicus 0.17

Methanobacterium thermo 0.15

Thermotoga maritima 0.13

Methanococcus jannasc 0.13

Archaeoglobus fulgidus 0.11

Mycoplasma genitalium 0.06

Synechocystis PCC6803 0.08

Ureaplasma urealyticum 0.07

Neisseria meningitidis 0.06

Mycobacterium tubercu 0.07

Rickettsia prowazekii 0.06

Haemophilus influenzae 0.05

Escherichia coli 0.05

Treponema pallidum 0.03

Helicobacter pylori 0.03

Bacillus subtilis 0.01

Genome f (S
-
S)

Pyro. aerophilum 0.44 104
°
C

Aero. pernix 0.40 100
°
C

Pyro. abyssi 0.31 102
°
C

Pyro. horikoshii 0.28 102
°
C

Aqui. aeolicus 0.17 93
°
C

Meth. thermo 0.15 90
°
C

Blue = archaea

= thermophile

90
°
C

86
°
C

92
°
C


Mallick, Boutz, Eisenberg, and Yeates (2002). PNAS 99, 9679
-
9684

Archaeal branch

Eubacterial branch

Exploring disulfide
-
rich
hyperthermophiles


Find the sequences of glutaredoxin
-
like protein
in different organisms.



Investigate the glutaredoxin
-
like protein in those
disulfide
-
rich hyperthermophiles.



Goals
: differences between glutaredoxin
-
like
protein in hyperthermophiles and glutaredoxin
in organisms.



Intro



Comput.
Methods



Results &
Significance



Applications
& Future
Directions



Summary

Why glutaredoxin
-
like protein?


Only present among hyperthermophiles.


Operates in thiol
-
disulfide reaction via CXXC motif which either form
a disulfide (oxidized form) or a dithiol (reduced form).


Requires for many functions including electron and proton transport
to essential enzymes like ribonucleotide reductase.


Involves in formation of disulfide bonds in protein folding.

90
o

Prototypical fold:
E.coli
thioredoxin (
2TRX.pdb
)

Methods


The sequences used in this study were obtained from
the National Center for Biotechnology Information
(www.ncbi.nlm.nih.gov).



Obtain the control sequence of glutaredoxin (
E. coli)

to
find glutaredoxin
-
like protein.



Search for the glutaredoxin
-
like protein sequences of
hyperthermophilic archaea.



Using Sequence
-
Structure Mapping to identify potential
disulfide bonds.



Compare and analyze using multiple sequences
alignment program, such as ClustalW, T
-
Coffee, or
MSA.


Intro



Comput.
Methods



Results &
Significance



Applications
& Future
Directions



Summary

Results



ClustalW multiple sequences alignment of these glutaredoxin
-
like
proteins shows
two

CXXC motifs



Glutaredoxin
-
like protein has more than 85 amino acids.

Green = P. aerophilum

Black = Hyperthermophilic archeae

Blue = Bacteria


= CXXC motif

Two CXXC motifs

Results


Most organisms have 1 CXXC
motif in glutaredoxin.


Glutaredoxin
-
like protein has
two

redox
-
active CXXC motifs per
polypeptide.


Exception
:
P. aerophilum

has
only 1 CXXC motif.


Intro



Comput.
Methods



Results &
Significance



Applications
& Future
Directions



Summary

P. furiosus:

1A8L.pdb
,
Nat. Struct. Biol.

(1998),
5

(7)
602
-
611

1J08.pdb
, unpublished

P. horikoshii:

CXXC motifs

Limitation


Only 25 genomes.



Some glutaredoxin
-
like proteins
have not yet been sequenced.





Intro



Comput.
Methods



Results &
Significance



Applications
& Future
Directions



Summary

Applications and Further Studies:


How disulfide bonds involve in protein folding?



To identify disulfide
-
bonded protein
-
protein
interactions and networks.



To investigate the stability mechanisms by
disulfide bonds.


Intro



Comput.
Methods



Results &
Significance



Applications
& Future
Directions



Summary

Summary


Most of the hyperthermophiles have 2 CXXC motifs in
order to have abundant disulfide bonds.



The abundance of disulfide bonds appear to play a key
role in stabilizing protein at high temperature.



Intracellular disulfide bond is a characteristic of all
archaea or an adaptation to high temperature.



This study illustrates the power of integrating genomic
data with protein structure and function to illuminate
the chemistry and biology of unusual organisms.


Intro



Comput.
Methods



Results &
Significance



Applications
& Future
Directions



Summary

Acknowledgements

Yeates Lab

Dr. Todd Yeates

Morgan Beeby

Everyone else at Yeates lab


CalState LA

Research mentors

SoCalBSI Program

SoCalBSI interns


Support

National Science Foundation (NSF)

National Institutes of Health (NIH).

UCLA
-
DOE Center for Genomics and Proteomics