Challenges and Methods in Transmembrane Protein Structure Determination

Connie JefferyUniversity 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 thickHydrophobic 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 - CFTRMultidrug resistance proteins

7TM receptors - G protein-linked receptors

Structure solution #2Beta-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, RhodopsinPhotosynthetic reaction centers

PorinsLight harvesting complexes

Potassium channelsChloride channels

AquaporinTransporters

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 Proteins2. General Topologies3. Methods and Challenges

a. Overexpressionb. Purificationc. Crystallization

4. Jeffery Lab Research Interests

Expression of TM ProteinsProblems:

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 timeamount 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:

LipopeptidesLipopeptides = 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 ProteinsProblem: 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 areaNeed monoclonal Abs, and usually use fragmentCrystal contacts often between Abs

Additional Method for Crystallization of TM Proteins: Cubic lipid phases

Landau & Rosenbusch, PNAS 93:14532-14535Nollert 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 FamilyThree 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.

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Acknowledgements

UICDr. Joseph OrgelDiana Arsenieva

Ji Hyun LeeForum BhattKathy ChangVishal PatelBong Bae

Vidya MadhavanRyo Kawamura

Tea Boci

Financial SupportUIC Campus Research Board

UIC Cancer Center/American Cancer Society

Cystic Fibrosis Foundation

American Heart Association

American Cancer Society

NSF

Society for Biomolecular Sciences