challenges and methods in transmembrane protein structure determination connie jeffery university of...
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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
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.***
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
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
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.
Graduate Studies in Biology
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
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