Structural Biology of Membrane Proteins
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Structural Biology of Membrane Proteins
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Overview
About the Author
Reinhard Grisshammer has studied the structure and function of G-protein-coupled receptors during the past 15 years, establishing a bacterial expression system for production of these eukaryotic membrane proteins, and developing a large-scale purification procedure for obtaining milligram quantities of homogeneous, functional receptors for structural studies.
Susan Buchanan has studied bacterial outer membrane proteins that transport iron and other small or large molecules during the past 10 years, developing methods for high level expression, purification, and crystallization, followed by structure determination by X-ray crystallography.
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Structural Biology of Membrane Proteins
By Reinhard Grisshammer, Susan K. Buchanan
The Royal Society of Chemistry
Copyright © 2006 The Royal Society of ChemistryAll rights reserved.
ISBN: 978-0-85404-361-3
CHAPTER 1
Refolding of G-Protein-Coupled Receptors
JEAN-LOUIS BANÈRES
UMR 5074 CNRS, "Chimie Biomoléculaire et Interactions Biologiques", Faculté de Pharmacie, 15 av. Ch. Flahault, BP14491, 34093 Montpellier Cedex 05, France
1 Introduction
G-protein-coupled receptors (GPCRs) are transmembrane receptors that are involved in the recognition and transduction of messages as diverse as light, C2+ ions, odor-ants, small molecules (including amino-acids, nucleotides, peptides) and proteins. Although different classes of receptors have been described, they all share a common structural motif composed of seven α-helices spanning the plasma membrane.
Although significant progress has been made within the last few years in dissecting GPCR-mediated signal transduction pathways, understanding the mechanisms underlying ligand recognition and signal transduction across the membrane has been hampered by the lack of information at the molecular level. This is largely due to the low abundance of most GPCRs in cellular membranes. Furthermore, few expression systems have proven satisfactory for producing these receptors in a functional state and sufficient yields." Structural information on the GPCR family is therefore very sparse, with the exception of rhodopsin for which X-ray and electron diffraction data have been obtained.
Recombinant expression has been one of the major bottlenecks in structural biology of GPCRs. One of the most widely used expression systems for structural biology is Escherichia coli However, in general, bacterial expression has been hampered by the relatively low yields of GPCRs owing to the toxic effects caused by these 7TM receptors when inserted into the bacterial membrane. To circumvent this toxicity problem, GPCRs can be directed to bacterial inclusion bodies. This leads to high expression levels of the receptor (in the range of 10-50 mg of protein per liter of bacterial culture). However, the highly expressed recombinant receptors are inactive and require refolding into a functional form. This explains why intensive work has been developed during the last years in analyzing the refolding of GPCRs and in devising efficient strategies for refolding receptors solubilized under denaturing conditions.
Understanding the basic principles of membrane protein folding is also of great interest in a fundamental perspective. Many studies have been dedicated in the past years to understand the trafficking of GPCRs. The quality control process in the endoplasmic reticulum involves a variety of mechanisms. These mechanisms ensure that only correctly folded proteins are directed to the plasma membrane. Despite this stringent quality control mechanism, gain- or loss-of-function mutations affecting protein folding in the endoplasmic reticulum, which have been described, can have profound effects on the health of an organism. Understanding the molecular mechanisms of protein folding could therefore help in correcting the structural abnormalities associated with misfolded receptors.
There are essentially two main types of membrane-spanning structures: trans-membrane α-helices and β-barrels. The latter appear to be limited to outer membrane proteins. The present work will focus on the refolding of α-helical membrane proteins. This is the most general case and the most interesting from a pharmacological point of view. It applies, in particular, to the GPCR family.
Investigating the in vitro refolding of membrane proteins is a difficult task, in particular, due to the hydrophobic nature of integral membrane proteins. Indeed, to work with isolated membrane proteins, one has to manipulate refolding solvents, generally composed of detergents or detergent/lipid mixtures, which poorly mimic the natural membranes. Nevertheless, biophysical studies on model systems have begun to provide a sound physical basis for membrane protein folding.
2 Refolding of Membrane Proteins
As stated above, one of the greatest problems in setting up conditions for studying a membrane protein in vitro arises from the relative instability of these proteins in detergent solutions. This problem, associated with the low abundance of most GPCRs in cellular membranes, explains the limited number of examples of refolding studies with GPCRs. Most of the biophysical studies on α-helical membrane protein folding have been carried out with bacteriorhodopsin as a model system, since it is a membrane protein that can be purified in high yields and is relatively stable in solution. Moreover, high-resolution structures of bacteriorhodopsin are available that help to understand the folding of this protein on a molecular basis. Bacteriorhodopsin functions as a light-driven proton pump in the purple membrane of the archaebacterium Halobacterium salinarium. As is the case with GPCRs, it possesses seven trans-membrane α-helical segments. Although it may not be fully representative of GPCRs at the folding level, it is nevertheless a good model system to gain a better understanding of receptor folding. Some general rules for a-helical membrane protein folding have been inferred from folding/unfolding studies with bacteriorhodopsin. In particular, a two-stage model has been proposed for the folding of these proteins that decomposes this process. First, individually stable transmembrane helices form and then these helices pack to form a functional protein (Figure 1).
The first step in the two-stage model thus involves the formation of the helical segments, and transmembrane helices display some characteristic features. In general, they are largely hydrophobic sequences that include a limited number of polar or potentially charged groups. In contrast to what is observed in globular proteins, prolines and glycines are also often found in membrane helices. The bending of a helical segment induced by a proline residue could be an important feature for membrane protein function. This is, for example, the case for the GPCR rhodopsin, where kink-inducing proline residues are found at key positions of the three-dimensional structure. Individual transmembrane helical structures are stable essentially because of hydrophobic effects and hydrogen bonds that are strong in the low dielectric environment of the membrane. Many helices found in membrane proteins are likely to be considered as stable folding units. In agreement with this view, individual helical segments or fragments of bacteriorhodopsin or GPCRs, containing only a reduced number of helices, can reach a stable fold in a membrane mimicking environment (see below).
If individual helices are formed in response to main-chain hydrogen bonding and hydrophobic effects, other interactions are likely to be involved in their assembly in the second stage of refolding. An important factor is certainly the way that the helices fit together, guided by Van der Waals interactions and side-chain rotamers. Another factor that is likely to influence the assembly of the transmembrane helices is the lipid environment. Besides its general role as a solvent, the lipid may also stabilize membrane proteins through specific interactions. Indeed, there are many examples of specific associations of individual lipids or of classes of lipids with membrane proteins. The interhelical loops can also have a role in the formation of helix/helix contacts, although the fact that proteins can in some cases assemble from fragments to form functional species suggests that the constraint induced by the loops may not be essential for folding. The loops could nevertheless promote folding events that bring transmembrane helices together. In agreement with this view, in the case of bacteriorhodopsin, the specific conformation of all the protein loops, except the DE loop, contributes to protein stability and is required for the correct folding and function of the protein.
In closing this part, although it may not fully represent the folding process of GPCRs, the two-stage model is certainly a good working basis for a better understanding of the folding of membrane proteins. A three stage model has also been proposed that, in addition to the two first steps, introduces a third step corresponding to ligand binding, folding of extramembranous loops, insertion of peripheral domains and formation of quaternary structures.
3 In Vitro Protein Refolding
Inclusion body production is a recurrent theme in recombinant protein technology. Refolding of inclusion bodies consists first in solubilizing the protein under denaturing conditions and then refolding is initiated by the removal of the denaturing agent. This can be done by dialysis or dilution. Protein folding has also been achieved by binding the protein to a chromatographic resin in the unfolded state and subsequent washing with an appropriate buffer that contains no denaturant. Affinity resins, such as Ni-NTA Sepharose or heparin Sepharose have been used for poly-histidine-tagged or poly-arginine-tagged proteins, respectively. Compared to folding by dialysis or rapid dilution, this method has the main advantage of preventing aggregation due to intermolecular interaction of partly folded protein species. However, an interference of the chromatographic support with the folding protein molecule may be detrimental, especially in the case of the highly hydrophobic membrane proteins, possibly causing precipitation of the protein on the matrix.
The efficiency of refolding depends on the competition between protein refolding and aggregation. One of the main difficulties in refolding membrane proteins is thus to find conditions that favor refolding over aggregation. A delicate balance must be reached between too harsh or too mild environments. Protein folding screens for identification of optimal folding conditions have been developed during the past years to screen different factors that may influence globular protein refolding." In a general manner, this consists in screening multiple conditions, in which different parameters (additives, pH, salt and protein concentration) are altered. Such folding screens can be adapted to integral membrane proteins, keeping in mind that one of the most crucial parameters to test will be the nature of the detergent and/or deter-gent/lipid mixture. This is indeed likely to be the factor with the most dramatic effect on membrane protein refolding efficiency.
4 GPCR In Vitro Refolding
4.1 Resolubilization from Inclusion Bodies
In most cases, refolding of GPCRs has been carried out with material recovered from bacterial inclusion bodies. As far as expression in E. coli is considered, it seems that there is no general strategy to be used for the efficient accumulation of GPCRs in inclusion bodies, even if some rules have been inferred from studies with different receptors. In some cases, the receptor simply fused to a T7 tag was efficiently expressed in E. coli. This is the case, for example, for the leukotriene B4 receptor BLT1 or, more recently, for the V2 vasopressin receptor. However, it must be noted that in the case of BLT1, protein expression levels dramatically vary from one clone to another. In other cases, fusion of the receptor to a protein partner was absolutely required for its expression. Different partners, such as glutathione S-transferase (GST) or ketosteroid isomerase (KSI) have been used. It must be emphasized again that no system allowing the expression of "all" GPCRs in inclusion bodies has been described so far. For example, no expression was observed with the 5-HT4a receptor fused to GST, whereas this receptor was efficiently produced when fused to KSI. Similarly, in our hands, among all the receptors we tested, KSI fusions gave inclusion bodies only with the 5-HT4a receptor. The best approach may therefore be to test different fusion partners and then quantify the expression levels.
Before starting the refolding, the inclusion body material has to be solubilized. Globular proteins in inclusion bodies can be solubilized in the presence of high concentrations of chaotropic agents, such as guanidinium hydrochlorideor urea. In contrast, aggregated membrane proteins require detergents (or organic solvents) for efficient solubilization, due to the predominance of hydrophobic effects in the aggregated material. Usually, SDS is used as a strong denaturing detergent. However, it must be kept in mind that, in SDS, helical membrane proteins, such as bacteriorhodopsin or GPCRs are not totally unfolded, but they can retain a significant amount of secondary structure. Indeed, sequence regions that, in the folded structure, form transmembrane helices tend to locally adopt an α-helical structure even in SDS. Bacteriorhodopsin, for example, is about 40–45% helical in SDS. The 5-HT4a receptor is also 30–35% helical in SDS-containing buffers (Banéres, unpublished data). The SDS-solubilized starting point for refolding is thus not to be considered as a fully unfolded state but rather as a partially folded state, as far as the secondary structure is concerned.
4.2 Refolding
As stated above, "unfolded" membrane proteins are first solubilized in harsh detergents, such as SDS or lauroylsarcosine. Refolding will then consist in replacing this denaturing detergent by a detergent that will stabilize the three-dimensional fold of the membrane protein. Under these conditions, based on the two-stage model, regions that have a propensity to fold will do so and then interactions between protein segments will appear. Those can be intramolecular, leading to refolding or intermolecular, leading to aggregation. One of the main challenges in refolding membrane proteins is therefore to find conditions, in particular, the nature of the detergent environment, which will favor the intramolecular over intermolecular contacts, and therefore refolding over aggregation. This implies finding the right balance between harsh and mild environments. For GPCRs, however, in the absence of extensive examples of successful receptor refolding, it is difficult to infer a general rule for the lipid and detergent requirements for maximal refolding efficiency.
Two different refolding studies have been reported so far for GPCRs. In the first case, the refolding involved, as described for bacteriorhodopsin, peptides encompassing one or several transmembrane domains obtained either by peptide synthesis, restricted protease digestion of receptors or bacterial expression in inclusion bodies. In the second case, the intact receptor was produced in bacterial inclusion bodies and then refolded in vitro.
4.3 Refolding of GPCR Fragments
We will not consider here the refolding of the extracellular ectodomains of GPCRs that has been described for several receptors, since these domains behave as typical globular proteins. We will focus here only on the transmembrane regions of GPCRs. Several papers reporting the refolding of fragments containing some of the transmembrane helices of GPCRs have been published so far. These fragments range from a single transmembrane domain to several transmembrane helices. As predicted by the two-stage model for membrane protein refolding, these fragments form folded domains when transferred from denaturing conditions to a milder environment. This suggests that GPCR transmembrane helices can also be considered as individual folding units. Besides their interest for a better understanding of the molecular processes involved in receptor folding, these studies indicate that the study of receptor fragments could be an alternative to the analysis of the structural properties of GPCRs. We will provide here two recent examples to illustrate this aspect of GPCR refolding.
(Continues...)
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Table of Contents
Part One: Expression and Purification of Membrane Proteins; Chapter 1: Refolding of G-protein-coupled receptors; 1: Introduction; 2: Refolding of membrane proteins; 3: In vitro protein refolding; 4: GPCR in vitro refolding; 5: Conclusion; 6: References; Chapter 2: Expression of genes encoding eukaryotic membrane proteins in mammalian cells; 1: Introduction; 2: Mammalian cell hosts and gene expression vectors; 3: Delivery and maintenance of expression vectors in mammalian host cells; 4: Use of HEK293S stable cell lines for high-level expression of eukaryotic membrane proteins; 5: Large-scale growth of HEK293S stable cell lines in suspension culture using a bioreactor and inducible gene expression; 6: Preparation of eukaryotic membrane proteins containing simple N-glycans; 7: Outlook for use of HEK293S tetracycline-inducible cell lines for large-scale preparation of other eukaryotic membrane proteins; Chapter 3: Expression of recombinant G-protein-coupled receptors for structural biology; 1: Introduction; 2: Expression of recombinant GPCRs; 3: Conclusions; 4: References; Chapter 4: The purification of G-protein-coupled receptors for crystallization; 1: Introduction; 2: Heterogeneity of overexpressed receptors; 3: Membrane fractionation, solubilization and detergent selection; 4: Purification; 5: Final quality control, monitoring protein stability, aggregational state, lipid and bound detergent; 6: Conclusions; Chapter 5: An introduction to detergents and their use in membrane protein studies; 1: Introduction; 2: Physical properties of detergents used in membrane protein studies; 3: Extraction and purification procedure using common detergents; 4: Use of detergents in membrane protein crystallization; 5: Conclusion; 6: References; Part Two: Methods for Structural Characterization of Membrane Proteins; Chapter 6: Solution NMR approaches to the structure and dynamics of integral membrane proteins; 1: Introduction; 2: Protein production and optimization for NMR studies; 3: NMR methodology for the study of integral membrane proteins; 4: Solution NMR structures of helical integral membrane proteins; 5: Solution NMR Structures of δ-barrel membrane proteins; 6: Solution NMR characterization of membrane protein dynamics; 7: Future directions; 8: References; Chapter 7: Membrane proteins studied by solid-state NMR; 1: Introduction; 2: Sample preparation and methodology; 3: Applications; 4: Conclusions; 5: References; Chapter 8: Assessing structure and dynamics of native membrane proteins; 1: Introduction; 2: Assembly of 2D crystals; 3: Electron microscopy; 4: Atomic force microscopy; 5: Conclusion and perspectives; Chapter 9: State-of-the-art methods in electron microscopy, including single particle analysis; 1: Introduction; 2: Sample Preparation; 3: Low Dose Microscopy; 4: Applications of CryoEM; 5: Examples; 6: Conclusion; Chapter 10: Atomic resolution structures of integral membrane proteins using cubic lipid phase crystallization; 1: Introduction; 2: Membrane protein crystals & crystallization; 3: Advantages of structures in a native setting at high resolution; 4: Conclusions; 5: References; Part Three: New Membrane Protein Structures; Chapter 11: Aquaporins: Integral membrane channel proteins; 1: Introduction; 2: Aquaporin channel proton exclusion barrier; 3: Selectivity in the aquaporin family; 4: Permeation by substances other than water and glycerol; 5: Aquaporin monomer associations and their functional implications; 6: References; Chapter 12: Gas channels for ammonia; 1: Introduction; 2: The structure of Ammonia Channel; 3: Reconstituted into liposomes AmtB acts as a channel that conducts NH3; 4: The Mechanism of conduction; 5: The Rh proteins; 6: Comparison with AQPs; 7: Comparison with K+ channel; 8: Acknowledgement; 9: References; Chapter 13: Channels in the outer membrane of Mycobacter; 1: Introduction; 2: Structure determination; 3: Structure description; 4: The outer membrane; 5: Conclusion; 6: References; Chapter 14: The structure of the SecY protein translocation channel; 1: Abstract; 2: Introduction; 3: Structure determination of the SecY complex by electron cryo-microscopy; 4: Determination of the X-ray crystal structure of the SecY complex; 5: Description of the structure of the SecY complex; 6: Post-translational translocation in bacteria; 7: Conclusions and outlook; 8: References; Chapter 15: Structure and function of the translocator domain of bacterial autotransporters; 1: Introduction; 2: The NalP autotransporter; 3: The translocator domain of autotransporters; 4: Purification and in vitro folding of the NalP translocator domain; 5: The structure of the NalP translocator domain; 6: Comparison of the NalP translocator domain to other translocator domains and to TolC; 7: The autotransporter secretion mechanism; Chapter 16: X-ray crystallographic structures of sarcoplasmic reticulum Ca2+-ATPase at the atomic level; 1: Introduction; 2: The transport scheme and thermodynamics of Ca2+ transport; 3: Overall structure of Ca2+-ATPase; 4: Transport models; 5: Initialization of the cycle - phosphorylation and calcium ion occlusion; 6: The dephosphorylation step and proton counter transport; 7: Getting Ca2+ in and out of the membrane; 8: Compact versus open conformations of SERCA; 9: Conclusions; Chapter 17: Comparison of the multidrug transporter EmrE structures determined by electron cryo-microscopy and X-ray crystallography; 1: Introduction; 2: The oligomeric state of EmrE; 3: Transport activity of EmrE; 4: Structure of EmrE determined by electron cryomicroscopy; 5: Comparison of the EmrE structure determined by electron crystallography with a 3.8 + resolution structure determined by X-ray crystallography; 6: Conclusion; 7: References; Chapter 18: Structure of photosystems I and II; 1: Introduction to oxygenic photosynthesis; 2: Photosystem ii; 3: Photosystem I; 4: Conclusion and outlook; Chapter 19: Glutamate receptor ion channels - Structural insights into molecular mechanism; 1: Introduction; 2: Studies of the Ligand-binding Domain; 3: The Functional Architecture of a Glutamate Receptor Ion Channel; 4: A Working Model of AMPA Receptor Function; 5: Open Questions; 6: References; Chapter 20: The mitochondrial ADP/ATP carrier; 1: Introduction; 2: Mitochondrial carriers and ADP/ATP carrier; 3: Crystallization; 4: Diffraction, phasing and model building; 5: Structure analysis; 6: Functional implications; 7: Future developments and conclusions;What People are Saying About This
...an up-to-date account of the state of the art in this field....a snapshot of the state of the art in the structural biology of membrane proteins. This book fills a gap and can serve both the membrane-protein researcher who is interested in other methods as well as other scientists who wish to get a general overview of the current state of membrane-protein structural biology. A very well presented and informative account of structural biology of membrane proteins.