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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

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Challenges and Methods in Transmembrane Protein Structure Determination Connie JefferyUniversity of Illinois at Chicagocjeffery@uic.edu

outline
Outline

1. Importance of Transmembrane Proteins

2. General Topologies

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

4. Jeffery Lab Research Interests

transmembrane proteins
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 proteins5
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!

outline6
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
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
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
Structure Solution #1: Hydrophobic alpha-helix
  • Satisfies polypeptide backbone hydrogen bonding
  • Hydrophobic sidechains face outward into lipids
examples of helix bundle tm proteins
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
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
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
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
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
Difficult and risky, but still possible:TM Proteins of Known Structure

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.***

Great summary and resource:

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

outline17
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
Expression of TM Proteins

Problems:

Low natural expression levels

Don’t always overexpress in recombinant systems

Formation of Inclusion bodies

expression of tm proteins19
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
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 proteins21
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
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
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
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
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

additional method for crystallization of tm proteins co crystallization with antibodies
Additional Method for Crystallization of TM Proteins: Co-crystallization with Antibodies
  • Increase hydrophilic surface area
  • Need monoclonal Abs, and usually use fragment
  • Crystal contacts often between Abs

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

additional method for crystallization of tm proteins cubic lipid phases
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
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
outline31
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
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
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
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 studies35
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
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 interests37
Jeffery Lab Research Interests
  • Proteomics-style systemmatic study of TM protein expression
  • Structure and Function of Multidrug Transporters
multidrug resistance
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
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
RND Protein Family

Some structural information is available for individual components

Three components:Outer membrane channel

+

Periplasmic protein

+

Inner Membrane transporter

Reference for figure:

rnd mdr family
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
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
slide43

University of Illinois at Chicago

Graduate Studies in Biology

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.

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

Acknowledgements