Transmembrane Proteins - ACA Summer School in Macromolecular ...

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Dec 6, 2012 (4 years and 9 months ago)

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Challenges and Methods in
Transmembrane Protein

Structure Determination


Connie Jeffery

University of Illinois at Chicago

cjeffery@uic.edu

Outline

1. Importance of Transmembrane Proteins

2. General Topologies

3. Methods (and challenges) for Structural
Studies of TM Proteins

4. Jeffery Lab Research Interests




Eukaryotic cells have many membranes

Transmembrane Proteins


Cellular roles include:


Communication between cells


Communications between organelles and cytosol


Ion transport, Nutrient transport

Links to extracellular matrix

Receptors for viruses

Connections for cytoskeleton


Over 25% of proteins in complete genomes.


Key roles in diabetes, hypertension, depression,
arthritis, cancer, and many other common diseases.


Targets for over 75% of pharmaceuticals.


Transmembrane Proteins


Cellular roles include:


Communication between cells


Communications between organelles and cytosol


Ion transport, Nutrient transport

Links to extracellular matrix

Receptors for viruses

Connections for cytoskeleton


Over 25% of proteins in complete genomes.


Key roles in diabetes, hypertension, depression,
arthritis, cancer, and many other common diseases.


Targets for over 75% of pharmaceuticals.


However, very few TM protein structures have been solved!

Outline

1. Importance of Transmembrane Proteins

2. General Topologies

3. Methods (and challenges) for Structural
Studies of TM Proteins

4. Jeffery Lab Research Interests




Biological Membrane = Lipid Bilayer

Approximately 30Å thick

Hydrophobic core + Hydrophilic or charged headgroups

Mixture of lipids that vary in type of head groups, lengths of acyl chains,

number of double bonds

(Some membranes also contain cholesterol)

Membrane Bilayer with Proteins

In order to be stable in this environment, a polypeptide chain needs to

(1) contain a lot of amino acids with hydrophobic sidechains, and

(2) fold up to satisfy backbone H
-
bond propensity
-

How?

Structure Solution #1:
Hydrophobic alpha
-
helix


Satisfies polypeptide
backbone hydrogen
bonding


Hydrophobic
sidechains face
outward into lipids

Examples of Helix Bundle TM Proteins

PDB = 1QHJ

PDB = 1RRC

Single helix or helical bundles

(> 90% of TM proteins)

Examples: Human growth hormone receptor, Insulin receptor

ATP binding cassette family
-

CFTR

Multidrug resistance proteins

7TM receptors
-

G protein
-
linked receptors

Structure solution #2

Beta
-
barrel


Beta sheet satisfies
backbone hydrogen
bonds between
strands


Wrap sheet around
into barrel shape


Sidechains on the
outside of the barrel
are hydrophobic

Examples of Beta Barrel TM Proteins

PDB = 1EK9

PDB = 2POR

Beta barrels

-

in outer membrane of gram negative bacteria,

and some nonconstitutive membrane acting toxins

Examples: Porins

General Topologies of TM Proteins


Single helix or helical bundles

and
Beta barrels

Both topologies result in

hydrophobic surfaces facing acyl chains of lipids

Part protruding from membrane can be a very short sequence (a few
amino acids), a loop, or large, independently folding domains

Presence of Hydrophobic TM
Domain can result in:

Low levels of expression

Difficulties in solubilization

Difficulties in crystallization


Attempting crystallization and structure
solution of transmembrane proteins is
considered difficult and risky.

Difficult and risky, but still possible:

TM Proteins of Known Structure

Great summary and resource:

http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html

Bacteriorhodopsin, Rhodopsin

Photosynthetic reaction centers

Porins

Light harvesting complexes

Potassium channels

Chloride channels

Aquaporin

Transporters

Etc.

**Although few in number, each of these structures
have been important for addressing key functions.***



Steps in X
-
ray Crystallography

Outline

1. Importance of Transmembrane Proteins

2. General Topologies

3.
Methods and Challenges


a. Overexpression


b. Purification


c. Crystallization

4. Jeffery Lab Research Interests




Expression of TM Proteins

Problems:


Low natural expression levels



Don’t always overexpress in recombinant systems


Formation of Inclusion bodies

Expression of TM Proteins

Potential Solutions (also can help in studies of soluble proteins):



Find cell type that naturally expresses a great deal of the protein



Scale up culture sizes



Change growth conditions
-



temperature
-

15
°
C, 30
°
C, 37
°
C, etc.


media


inducing time


amount of inducing agent



Change expression vectors



Change strain or even species of expression host



Try many members of a protein family
-

related proteins

and/or proteins from different species:

Methods for Solubilization and
Purification of TM Proteins

Problem: Hydrophobic domains
tend to aggregate when taken
out of the lipid bilayer
-

result in
sticky precipitant of unfolded
proteins


Solution: Include mild detergent(s)
in purification steps
-

will mask
the hydrophobic regions and
help solubilize the protein




Methods for Solubilization and
Purification of TM Proteins




Note: Trial and error needed to find good detergent
that keeps protein folded and active


Might try many detergents with different head groups

And acyl chain lengths.


Beta
-
octylglucoside = example of a common mild
detergent used with studies of membrane proteins

Alternative Reagents for
Solubilization of TM Proteins

Design, synthesis, and use of:


More kinds of detergents


Detergents with novel structures (example from Prot. Science 2000,
9:2518
-
2527)

Alternative Reagent for
Solubilization of TM Proteins:

Lipopeptides

Lipopeptides = Novel detergent/peptide hybrids


(see McGregor
et al.
,
Nature Biotechnology

2003, 21:171
-
176)

(Figures from McGregor
et al.
,
Nature Biotechnology

2003, 21:171
-
176)

Alternative Reagent for
Solubilization of TM Proteins:
Nanodiscs


From Steven Sligar lab at
UIUC.


Goal is to put individual
TM protein in environment
that mimics lipid bilayer
better than a micelle


Nanodiscs contain small
phospholipid bilayer
wrapped by membrane
scaffold protein

Figure from pamphlet from office of

technology management, UIUC

Crystallization of TM Proteins

Problem: Hydrophobic
domains tend to aggregate
when taken out of the lipid
bilayer
-

result in sticky
precipitant of unfolded
proteins


Solution: Include mild
detergent(s) in
crystallization steps
-

will
mask the hydrophobic
regions and help solubilize
the protein, special screens
developed for TM proteins




Note: Probably need to modify lipids and/or detergents plus
modifying other components of crystallization solution

Crystallizing Proteins

Additional Method for Crystallization

of TM Proteins: Co
-
crystallization with
Antibodies




Figure modified from Hunte and Michel, Current Opinion Structural Biology, 2002, 12:503
-
508.


Increase hydrophilic
surface area


Need monoclonal Abs,
and usually use fragment


Crystal contacts often
between Abs

Additional Method for Crystallization

of TM Proteins: Cubic lipid phases




Landau & Rosenbusch,
PNAS 93:14532
-
14535

Nollert et al., Methods
Enz. 343:183
-
199.



3
-
dimensional lipid bilayer structure that forms in
mixtures of certain lipids and water (i.e. monoolein, PNAS
(1996) 93, pp. 14532
-
14535).


TM protein is found crossing bilayer and can interact
with other copies of the protein at various angles.


Alternative solution for Crystallization

of TM Proteins

: Extramembranous
Domains alone

PDB = 2LIG



Some proteins: regions outside the bilayer are
globular domains that contain the key enzymatic
or binding functions.



Study these domains separate from the
membrane spanning domain (using recombinant
DNA techniques)


The isolated domain can often be treated like a
soluble protein.

--
>


Examples
-

aspartate receptor, human growth hormone receptor

Steps in X
-
ray Crystallography

Outline

1. Importance of Transmembrane Proteins

2. General Topologies

3. Methods (and challenges) for Structural
Studies of TM Proteins

4. Jeffery Lab Research Interests




Jeffery Lab Research Interests


Proteomics
-
style systematic study of TM protein expression


Structure and Function of Multidrug Transporters

A proteomics level approach to

TM protein studies

Selection of proteins with a variety of

physical characteristics and functions
-

Begin with study of expression and solubilization methods.

Cystic Fibrosis


Lethal genetic disease


1 in 20 caucasions is a carrier


1 in 2000 live births


Affects lungs, pancreas, sweat
ducts, reproductive organs


Thick mucus secretions


Caused by mutations in the
CFTR protein


Low life expectancy due in part
to recurrent serious lung
infections with
P. aeruginosa,
a
multidrug resistance
opportunistic bacterium.

A proteomics level approach to

TM protein studies

Clone >100 target

TM proteins into
similar vectors.

Use constructs to
test methods of
expression,
solubilization ,
purification, and
crystallization.

Figure modified from Gateway cloning system information from Invitrogen.

To be evaluated:


Do expression and membrane localization
correlate with



Physical features or function of the protein?



Expression conditions? (including temperature,
tags, vectors, strains, etc.)


Jeffery Lab Research Interests


Proteomics
-
style systemmatic study of TM protein expression


Structure and Function of Multidrug Transporters

Multidrug Resistance



Increasing problem in medicine: bacteria becoming
resistant to wide range of antibiotics



Caused by 5 major familes of transmembrane
transporters (RND, ABC, MATE, SMR, MFS)




Pump many kinds of antibiotics out of cell



Info about mechanisms of functions would be useful for


finding efflux inhibitors


finding novel antibiotics that aren’t pumped

MDRs of RND Protein Family

Three components:

Outer membrane channel + Periplasmic protein + Inner Membrane transporter

Somehow the proteins
work together to form a
complex that crosses
both membranes. The
drug is accepted from
the periplasm or inner
membrane and
transported through the
outer membrane. We
are working on
individual proteins and
complexes from
Pseudomonas
aeruginosa
.

RND Protein Family

Three components:

Outer membrane channel

+


Periplasmic protein


+

Inner Membrane transporter

Some structural
information is available for
individual components

Reference for figure:


RND MDR Family


Additional structures and biochemical/biophysical
characterization would help with:



How do the 3 protein components fit together?



How is proton motive force used to pump drugs?



How do inhibitors inhibit the pumps?



How do the different RND transporters select different
subsets of drugs?



What compounds (novel antibiotics) would escape
pumps?

Summary


Transmembrane Proteins play many important processes in
cellular processes in both health and disease


Two general type of tertiary structure are found to cross the
membranes: beta
-
barrels and alpha
-
helices


Structural Studies of TM Proteins are impeded by difficulties in
overexpression, purification and crystallization


However, the few dozen structures that have been determined
have provided key information about channels (gating,
selectivity, etc.), energetics, transport, and other
transmembrane processes


University of Illinois at Chicago

The Department of Biological
Sciences at UIC provides training
leading to the Ph.D. degree in
Molecular, Developmental and
Cellular Biology.

Full tuition waiver & competitive stipend available for qualified candidates.


For more information visit http://www.uic.edu/depts/bios.

Graduate Studies in Biology

Acknowledgements

UIC

Dr. Joseph Orgel

Diana Arsenieva

Ji Hyun Lee

Forum Bhatt

Kathy Chang

Vishal Patel

Bong Bae

Vidya Madhavan

Ryo Kawamura

Tea Boci



Financial Support

UIC Campus Research Board

UIC Cancer Center/American
Cancer Society

Cystic Fibrosis Foundation

American Heart Association

American Cancer Society

NSF

Society for Biomolecular
Sciences