Challenges and Methods in Transmembrane Protein Structure Determination Connie Jeffery University of Illinois at Chicago [e-mail]

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Challenges and Methods in Transmembrane Protein
Structure Determination Connie
JefferyUniversity of Illinois at
Chicagocjeffery_at_uic.edu
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Outline

• 1. Importance of Transmembrane Proteins
• 2. General Topologies
• 3. Methods (and challenges) for Structural
  Studies of TM Proteins
• 4. Jeffery Lab Research Interests

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Eukaryotic cells have many membranes
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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.

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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!
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Outline

• 1. Importance of Transmembrane Proteins
• 2. General Topologies
• 3. Methods (and challenges) for Structural
  Studies of TM Proteins
• 4. Jeffery Lab Research Interests

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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)
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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?
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Structure Solution 1 Hydrophobic alpha-helix

• Satisfies polypeptide backbone hydrogen bonding
• Hydrophobic sidechains face outward into lipids

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Examples of Helix Bundle TM Proteins
PDB 1QHJ
PDB 1RRC
Single helix or helical bundles (gt 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
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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

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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
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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

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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.

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Difficult and risky, but still possibleTM
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.u
ci.edu/Membrane_Proteins_xtal.html
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Steps in X-ray Crystallography
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Outline

• 1. Importance of Transmembrane Proteins
• 2. General Topologies
• 3. Methods and Challenges
• a. Overexpression
• b. Purification
• c. Crystallization
• 4. Jeffery Lab Research Interests

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Expression of TM Proteins
Problems Low natural expression levels
Dont always overexpress in recombinant
systems Formation of Inclusion bodies
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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 - 15C, 30C, 37C, 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

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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

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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-octylgl
ucoside example of a common mild detergent used
with studies of membrane proteins
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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, 92518-2527)

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Alternative Reagent for Solubilization of TM
ProteinsLipopeptides

• Lipopeptides Novel detergent/peptide hybrids
• (see McGregor et al., Nature Biotechnology 2003,
  21171-176)

(Figures from McGregor et al., Nature
Biotechnology 2003, 21171-176)
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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
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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
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Crystallizing Proteins
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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, 12503-508.
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Additional Method for Crystallization of TM
Proteins Cubic lipid phases
Landau Rosenbusch, PNAS 9314532-14535 Nollert
et al., Methods Enz. 343183-199.

• 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.

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Alternative solution for Crystallization of TM
Proteins Extramembranous Domains alone
--gt
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

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Steps in X-ray Crystallography
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Outline

• 1. Importance of Transmembrane Proteins
• 2. General Topologies
• 3. Methods (and challenges) for Structural
  Studies of TM Proteins
• 4. Jeffery Lab Research Interests

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Jeffery Lab Research Interests

• Proteomics-style systemmatic study of TM protein
  expression
• Structure and Function of Multidrug Transporters
• Folding of TM proteins (Determinants of Helical
  Packing)

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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.
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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.

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A proteomics level approach to TM protein studies
Clone gt100 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.
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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.)

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Jeffery Lab Research Interests

• Proteomics-style systemmatic study of TM protein
  expression
• Structure and Function of Multidrug Transporters
• Folding of TM proteins (Determinants of Helical
  Packing)

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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 inhibitors
• finding novel antibiotics that arent pumped

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MDRs of RND Protein Family
Three componentsOuter 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.
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RND Protein Family
Some structural information is available for
individual components
Three componentsOuter membrane channel
Periplasmic protein Inner Membrane transporter
Reference for figure
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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?
• What is path of drugs through protein?
• How do inhibitors inhibit the pumps?
• How do the different RND transporters select
  different subsets of drugs?
• What compounds (novel antibiotics) would escape
  pumps?

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Jeffery Lab Research Interests

• Proteomics-style systemmatic study of TM protein
  expression
• Structure and Function of Multidrug Transporters
• Folding of TM proteins (Determinants of Helical
  Packing)

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Protein Folding Problem

• How does a one-dimensional amino acid sequence
  determine a specific three-dimensional structure?
• Or
• How can we read the sequence and predict that
  structure?

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General Idea

• We know what an alpha-helix or a beta strand
  looks like, so
• (1) figure out which parts of the sequence are
  helices and which parts are strands
• (2) figure out how they pack together
• For soluble proteins, neither is well predicted.
• But for transmembrane proteins ...

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TM Protein Structure Prediction, Step 1

• For alpha-helical transmembrane proteins,
  hydropathy plot analysis provides a fairly
  accurate method to predict which amino acids form
  membrane-spanning helices

We can model the structure of an individual
alpha helix fairly accurately.
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TM Protein Structure Prediction, Step 2

• How do the helices pack in the membrane?
• There are several labs studying known protein
  structures to identify factors involved in
  determining how transmembrane helices pack
  together (specificity of interaction and packing
  motifs)
• Hydrogen bonds
• Hydrophobiciity
• Amino acids known to face the lumen of a channel
• Multiple sequence alignments
• Helix packing sequence motifs, etc.
• These kinds of information are then combined with
  protein docking and energy minimization programs
  to predict how the helices pack together.
• It is quite possible that studies of helical
  transmembrane proteins could lead to key
  information about the protein folding problem -
  how to predict protein structure from amino acid
  sequence

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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
• Analysis of helical transmembrane protein
  structures may lead to accurate predictions of
  protein structure from amino acid sequence for
  this type of protein

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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.
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Acknowledgements

• UIC
• Dr. Joseph Orgel
• Diana Arsenieva
• Ji Hyun Lee
• Forum Bhatt
• Kathy Chang
• Vishal Patel
• Bong Bae
• Vidya Madhavan
• Ryo Kawamura

• Financial Support
• UIC Campus Research Board
• UIC Cancer Center/American Cancer Society
• Cystic Fibrosis Foundation
• American Heart Association
• American Cancer Society