Archives of Biochemistry and Biophysics 564 (2014) 262–264
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Editorial
Membrane protein folding and stability
There’s no doubt: membrane proteins are difficult to work with. They don’t express to very high levels in heterologous systems. Once expressed they are often tricky to extract from the membrane fraction (and even more difficult to solubilize and fold if expressed as inclusion bodies), and have an annoying tendency to aggregate and become inactivated. Finally, getting them to fold reversibly in a bona fide membrane environment is a real challenge. But all these challenges are also in a sense exhilarating: we can learn so much about what really makes proteins ‘‘tick’’ by comparing their properties with those of water-soluble proteins – almost like wising up to our own understanding of life by studying alien life forms. So although membrane protein structures only make up around 2% of the protein structure data base (and roughly the same can be said about biophysical studies), all such insights are bound to make a real impact. The pace is picking up, and we can cheerfully estimate that we are only 30 years behind our water-soluble colleagues in structural determination. The present collection of articles, contributed by leading experts in the field, is an attempt to present some of the insights we already have and point out ways in which to direct our future explorations. McMorran, Brockwell and Radford start the collection with a review of the folding of outer membrane proteins [1]. They provide an excellent starting point with central concepts in protein folding obtained from two decades of intense studies of the folding of water-soluble folding; these include protein folding funnels and rugged energy landscapes as well as unified folding models such as the nucleation–condensation mechanism. Transferring these concepts to membrane proteins is challenged by the ability to recreate a membrane-like folding environment in vitro. However, the b-barrel outer membrane proteins (OMPs) are attractive folding models, as they can often be directly refolded from the completely unstructured state into phospholipid vesicles. This avoids the need for vesicle-disrupting detergents and allows studies of the effect of membrane composition and periplasmatic chaperones such as SurA, Skp, FkpA and others. These chaperones help OMPs transition the periplasm; the actual membrane insertion is assisted by the BAM complex, a fascinating apparatus containing an OMP and several accessory lipoproteins which can be reconstituted in vitro for more detailed mechanistic studies. There are intense efforts to elucidate how this complex assists OMP insertion and the results will undoubtedly help us understand better how the cell modulates the OMPs’ intrinsic physical–chemical properties to achieve rapid and efficient folding. After a brief overview of the appropriate techniques to use in OMP folding (ranging from SDS–PAGE and Trp fluorescence to NMR spectroscopy, all of which can be combined with protein engineering), the authors describe http://dx.doi.org/10.1016/j.abb.2014.10.014 0003-9861/Ó 2014 Elsevier Inc. All rights reserved.
the folding of two OMPs in detail. The 8-strand OmpA is the archetypal OMP which has been useful in developing methodology for OMP folding; however, the equally small (8-strand) PagP was the first OMP to be analyzed by protein engineering in the Radford group with an ingenious combination of phospholipid vesicles and high urea concentrations. In contrast to PagP, many OMPs suffer from hysteresis in folding, which means that they do not attain equilibrium between folded and unfolded states under normal experimental time scales. However, Karen Fleming’s group has which has spearheaded a systematic approach to identify reversible folding conditions into vesicles and thus lay the groundwork for more general studies of OMP folding and its coupling to the specific conditions in the cell. Combined with efforts to include chaperones the BAM complex and possibly even asymmetric membranes in in vitro studies, we can expect plenty of exciting advances in the OMP field over the next few years. In the article by Neumann, Klein, Otzen and Schneider, we switch to the other major class of membrane proteins, those composed mainly of a-helices [2]. There has been a steady progression in the complexity of these studies, starting with the classic singletransmembrane (TM) helix glycophorin which naturally dimerizes in a way that can be quantified both by transcription assays and SDS–PAGE; countless mutagenesis studies in vivo and in vitro have provided vital information about membrane helix packing motifs. Conceptually, the next step up is to understand what in practice drives folding of polytopic membrane proteins. The 7-TM protein bacteriorhodopsin was the first membrane protein to be reconstituted from a denatured state in vitro and has driven much of this development; its chromophore is both a blessing (to monitor the extent of native state formation) and a curse (since it complicates refolding kinetic studies). Reversible folding is also the holy grail for a-helical MPs; they are generally so hydrophobic (unlike OMPs) that they cannot be refolded from the urea- or guanidine-chloride denatured random coil state but have to be solubilized in SDS which induces an a-helical conformation that is more or less retained (though probably significantly rearranged) in the native state. A great advantage of using SDS as denaturant is that its denaturation potency can be systematically reduced by titrating in nonionic detergents such as dodecyl maltoside; although mixed micelles are undoubtedly not bona fide vesicles, this ‘‘tuning’’ of denaturation leads to fully reversible unfolding under equilibrium conditions and also opens up for comprehensive kinetic folding/ unfolding studies. So far we have protein engineering studies of the folding of three a-helical MPs in mixed SDS/DDM micelles, namely bacteriorhodopsin, the 4-TM DsbB and most recently the 6-TM membrane protease GlpG. These help to gradually build up
Editorial / Archives of Biochemistry and Biophysics 564 (2014) 262–264
a picture of a transition state which is closer to the SDS-denatured state than the native state in terms of compactness but clearly has defined regions of folded structure which are quite sharply segregated or even polarized. A novel observation is that GlpG appears to have a large region of ‘‘frustrated structure’’ surrounding a rather small folding nucleus while most of the C-terminal part is essentially unfolded; temptingly, these data highlight the N-terminus as the initiation of folding, just as we would expect in vivo. The next step is to connect this experimental insight with computational studies, which we expect will be the next great frontier in a-helical MP folding. Progressing from monomeric proteins, the final step is to understand how polytopic a-helical MPs associate to form complexes and oligomers. This work has been spearheaded by the trimeric diacyl glycerate kinase, the original model system for mixed micelle folding, where monomeric subunits fold (but not to completion) before trimerizing. The aquaglyceroporin GlpF is tetrameric in the native state, but can dissociate and unfold under mildly acidic conditions or in SDS; the latter case is completely reversible and proceeds through at least one moltenglobule-like dimer. In contrast, the potassium channel KcsA resists SDS and only dissociates and unfolds in trifluoroethanol, which is only completely reversible if small amounts of lipid is present. While the sugar transporter lacY is monomeric and unfolds reversibly to some extent in urea, another member of the major facilitator superfamily GalP is naturally trimeric, but can also be partially (and reversibly) unfolded in urea in the presence of DDM. This susceptibility to urea is probably due to the proteins’ low stability and high flexibility, which makes it easier to disrupt hydrophobic interactions holding the protein together. We summarize all these observations in 6 empirical rules, before moving on to a final section on how polytopic MPs oligomerize. Homo-oligomerization, often to form highly symmetric structures, is widespread among these MPs, probably caused by gene duplication. The degree of cooperativity between different monomeric units varies – in some cases only the oligomer is functional and is formed by cooperative folding while in other cases the monomers are the functional units. Cooperativity between monomers is best probed by genetic fusion of the protomers and specific introduction of mutations in one or more protomers. We categorize oligomerization in three classes; monomer folding and oligomerization may be tightly coupled (two-state or co-translational, typical for homo-oligomers such as channel proteins), they may constitute separate steps (three steps, most likely the case for hetero-oligomers) and finally oligomerization may be controlled by ligands or depend on cellular signals, as seen for G-protein coupled receptors. Oligomerization confers certain advantages: besides thermodynamic and biological (e.g. against proteases) stabilization of the protein, they provide an opportunity to evolve new functions, e.g. by modulating monomer–oligomer equilibria and opening up for allosteric regulation. This is summarized in 7 simple observational rules which point the way for future research in this area. After this guided tour of the mechanisms by which membrane proteins fold in membrane-like environments, the review by Heedeok Hong turns to the driving forces that make this process favorable – with a keen eye to the differences between water-soluble and membrane proteins [3]. The complexity of the membrane environment makes it clear that although the underlying physical principles of course are the same, the game rules determining MP stability differ fundamentally from those of water-soluble proteins. First of all, it might even be questioned whether folding is thermodynamically driven or under kinetic control. Reconstitution of bacteriorhodopsin from different denatured states and from fragments settled this in favor of thermodynamic control (at least in vitro) and led to Popot and Engelman’s seminal two-stage model for insertion and association of a-helical MPs. As discussed in the previous article, folding reversibility can be tested by mixed micelles and also
263
elaborated by combination with hydrogen–deuterium exchange, though it should be noted that the structure of the denatured state can vary with absolute SDS concentration. Therefore alternative approaches are needed: these include pulse-proteolysis and more recently steric trapping, in which unfolding allows binding of bulky tags like streptavidin; unlike conventional equilibrium denaturation (but like refolding kinetics), this allows direct measurement of stability at very low SDS concentrations and may help avoid incorrect linear extrapolations under these conditions – though data with GlpG folding kinetics indicate that linear correlations can exist at low SDS mole fractions. So what stabilizes these membrane proteins? The hydrophobic effect is a strong force in watersoluble protein folding, burying hydrophobic residues away from the hydrophilic solvent. However, this effect cannot be so pronounced in membrane proteins. Rather, efficient packing of buried residues, facilitated by small residues, optimizes (weak) van der Waals interactions in competition with lipid contacts – though there is apparently still room for improvement in many membrane proteins. Also, hydrogen bonding in MPs comes with a twist: close packing promotes CaHAO@C hydrogen bonds (particularly in dimerizing peptides), while more conventional hydrogen bonds seem to stabilize MPs to the same extent as water-soluble proteins. These effects may be modulated by penetrating water molecules, and changes in the hydrogen bonding pattern may even modulate overall helix architecture (kinked versus straight). Salt bridges between side chains with complementary charges are only weakly stabilizing in an aqueous environment while buried salt bridges do not stabilize water-soluble proteins more than their hydrophobic side chain replacements. There are no detailed studies of these interactions in a-helical MPs, and it may be difficult to extrapolate from data in OMPs due to major differences in polarity. Cation-p interactions, widespread in water-soluble proteins, go a step further in MPs where they mediate contact with the surrounding lipid and may rationalize the preponderance of aromatic residues in the interfacial region (which is particularly widespread in beta-barrel proteins). Cation–cation interactions only provide modest contributions to water-soluble protein stability, but their geometric specificity (favoring an edge-to-face configuration) likely contributes to specific interhelical contacts, though this remains to be explored in more detail – along with better modeling and mimicry of the specific membrane environment. Thus our understanding of forces specifically stabilizing MPs is probably only at the beginning of a steep learning curve which will be very exciting to follow. We should certainly not consider membrane protein studies to be an intractable problem. Jean-Luc Popot, whose two-stage model developed with Donald Engelman has done so much to stimulate thinking about membrane protein insertion and folding, is uniquely placed to prepare an overview of the current situation. He provides a veritable tour de force of the many different conditions used to fold and refold a-helical MPs and OMPs [4]. His compilation encompasses the 89 MPs (52 a-helical and 37 OMPs, of which 10 and 8, respectively, are oligomers) which had been reported to be folded in vitro by the end of 2013 (a number which far exceeded his initial expectations). The cases include both refolded (i.e. first denatured and then refolded) and folded (not previously native) proteins and span a number of different conditions. His article takes us on a historical survey of how folding experiments were important in developing our understanding of what a-helical MPs and OMPs ‘‘really are’’, how they behave in relation to the Anfinsen folding paradigm and the steps beyond simple dilution experiments (used for water-soluble proteins) to fold MPs. Interestingly, of the 42 MPs folded from inclusion bodies, around two thirds (27) are OMPs; the difficulty of folding a-helical MPs in this way may have stimulated the use of cell-free extracts for their expression, which is also a burgeoning field in membrane protein production. The table also lists more exotic denaturing and
264
Editorial / Archives of Biochemistry and Biophysics 564 (2014) 262–264
folding media such as sarkosyl and organic solvents and highlights helpers such as natural ligands as well as specific (non-annular) lipids. The actual mode of transfer from denaturing to folding conditions merits comments since it can be done in a variety of ways, though it generally appears that ‘‘faster is better’’. The great variety of ways to obtain folded MPs should not be seen as a bewildering obstacle but rather a versatile and helpful guide, which makes it more likely that you will eventually reach your goal and bring your own favorite MP to a safe (native) haven. As a final perspective on MP protein folding, Jean-Luc Popot continues with Jörg Kleinschmidt in a description of a synthetic group of amphipathic polymers called amphipols (APols) which hold great promise both in folding and stabilizing membrane proteins [5]. Like detergents, they contain both hydrophilic groups and hydrophobic chains, but as (short) polymers they interact less invasively with MPs than detergents do. Their polymeric nature means that they do not dissociate from MPs upon dilution (unless competed out by other detergents), which is a distinct advantage when handling MPs. Different APols are now available with different head-groups and thus different pH-sensitivities; recent additions remain water soluble even below pH 7 (the original APol, A8-35, has an ionizable carboxylate group and precipitates below pH 7). APols are superior to most conventional nonionic detergents (though not DDM) in resolubilizing bacteriorhodopsin and can replace SDS in a relatively straightforward manner. Bacteriorhodopsin is a robust protein, however. The real test is to see how APols perform with fragile G-protein coupled receptors and here APols are also promising; even without optimization, folding yields beyond 30% can be obtained for a variety of these receptors, heralding their potential in crystallization trials. Non-ionic APols (NAPols) also show promise, since their lack of charge reduces their unspecific binding to MPs and makes them superior to A8-35 in cell-free expression. APols are also good tools for folding of OMPs, since larger OMPs can be difficult to fold quantitatively; however, the 14-strand OMP FomA folds to 90% within 24 h with the expected gain of structure and functionality, while folding of OmpA into A8-35 shows a lower activation energy than folding into lipid bilayers. Experiments with new APols such as sulfonated
forms are also underway. Furthermore, once folded into APols, the MPs show much higher stability than in nonionic detergents, probably due to their tighter association as polymers. OmpA is less thermodynamically stable in A8-35 than in the nonionic detergent LDAO, but this may reflect the high pH of 10 required for folding, which will exacerbate electrostatic repulsion; nevertheless, it unfolds more slowly in A8-35 than in LDAO. These many intriguing observations can be explained as a combination of different phenomena: firstly, APols do not need to be present in excess (unlike detergents) and therefore do not constitute a large hydrophobic sink for e.g. monomerizing oligomeric MPs. However, even at concentrations comparable with detergents, APols stabilize MPs, since their polymeric structure and short hydrophobic chains makes it difficult for them to compete with protein/protein and protein/ lipid interactions. When they do bind MPs, they may do so by a ‘‘Gulliver effect’’, binding at multiple points and dampening or freezing MPs’ conformational fluctuations, as well as sterically preventing aggregation. These properties make APols a prominent member of the growing tool-box for membrane solubilization. I hope that you will enjoy reading these articles which highlight both problems and possibilities in this exciting field of research. I am very grateful to the many colleagues who have contributed to this and who have made it such a pleasure to edit this collection. References [1] L.M. McMorran, D.J. Brockwell, S.E. Radford, Arch. Biochem. Biophys. 564 (2014) 265–280. [2] J. Neumann, N. Klein, D.E. Otzen, D. Schneider, Arch. Biochem. Biophys. 564 (2014) 281–296. [3] H. Hong, Arch. Biochem. Biophys. 564 (2014) 297–313. [4] J.L. Popot, Arch. Biochem. Biophys. 564 (2014) 314–326. [5] J.H. Kleinschmidt, J.L. Popot, Arch. Biochem. Biophys. 564 (2014) 327–343.
Daniel Otzen Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Center for Insoluble Protein Structures (inSPIN), Aarhus University, Gustav Wieds Vej 14, DK – 8000 Aarhus C, Denmark Received 31 October 2014 Available online 1 November 2014
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Editorial
Membrane protein folding and stability
There’s no doubt: membrane proteins are difficult to work with. They don’t express to very high levels in heterologous systems. Once expressed they are often tricky to extract from the membrane fraction (and even more difficult to solubilize and fold if expressed as inclusion bodies), and have an annoying tendency to aggregate and become inactivated. Finally, getting them to fold reversibly in a bona fide membrane environment is a real challenge. But all these challenges are also in a sense exhilarating: we can learn so much about what really makes proteins ‘‘tick’’ by comparing their properties with those of water-soluble proteins – almost like wising up to our own understanding of life by studying alien life forms. So although membrane protein structures only make up around 2% of the protein structure data base (and roughly the same can be said about biophysical studies), all such insights are bound to make a real impact. The pace is picking up, and we can cheerfully estimate that we are only 30 years behind our water-soluble colleagues in structural determination. The present collection of articles, contributed by leading experts in the field, is an attempt to present some of the insights we already have and point out ways in which to direct our future explorations. McMorran, Brockwell and Radford start the collection with a review of the folding of outer membrane proteins [1]. They provide an excellent starting point with central concepts in protein folding obtained from two decades of intense studies of the folding of water-soluble folding; these include protein folding funnels and rugged energy landscapes as well as unified folding models such as the nucleation–condensation mechanism. Transferring these concepts to membrane proteins is challenged by the ability to recreate a membrane-like folding environment in vitro. However, the b-barrel outer membrane proteins (OMPs) are attractive folding models, as they can often be directly refolded from the completely unstructured state into phospholipid vesicles. This avoids the need for vesicle-disrupting detergents and allows studies of the effect of membrane composition and periplasmatic chaperones such as SurA, Skp, FkpA and others. These chaperones help OMPs transition the periplasm; the actual membrane insertion is assisted by the BAM complex, a fascinating apparatus containing an OMP and several accessory lipoproteins which can be reconstituted in vitro for more detailed mechanistic studies. There are intense efforts to elucidate how this complex assists OMP insertion and the results will undoubtedly help us understand better how the cell modulates the OMPs’ intrinsic physical–chemical properties to achieve rapid and efficient folding. After a brief overview of the appropriate techniques to use in OMP folding (ranging from SDS–PAGE and Trp fluorescence to NMR spectroscopy, all of which can be combined with protein engineering), the authors describe http://dx.doi.org/10.1016/j.abb.2014.10.014 0003-9861/Ó 2014 Elsevier Inc. All rights reserved.
the folding of two OMPs in detail. The 8-strand OmpA is the archetypal OMP which has been useful in developing methodology for OMP folding; however, the equally small (8-strand) PagP was the first OMP to be analyzed by protein engineering in the Radford group with an ingenious combination of phospholipid vesicles and high urea concentrations. In contrast to PagP, many OMPs suffer from hysteresis in folding, which means that they do not attain equilibrium between folded and unfolded states under normal experimental time scales. However, Karen Fleming’s group has which has spearheaded a systematic approach to identify reversible folding conditions into vesicles and thus lay the groundwork for more general studies of OMP folding and its coupling to the specific conditions in the cell. Combined with efforts to include chaperones the BAM complex and possibly even asymmetric membranes in in vitro studies, we can expect plenty of exciting advances in the OMP field over the next few years. In the article by Neumann, Klein, Otzen and Schneider, we switch to the other major class of membrane proteins, those composed mainly of a-helices [2]. There has been a steady progression in the complexity of these studies, starting with the classic singletransmembrane (TM) helix glycophorin which naturally dimerizes in a way that can be quantified both by transcription assays and SDS–PAGE; countless mutagenesis studies in vivo and in vitro have provided vital information about membrane helix packing motifs. Conceptually, the next step up is to understand what in practice drives folding of polytopic membrane proteins. The 7-TM protein bacteriorhodopsin was the first membrane protein to be reconstituted from a denatured state in vitro and has driven much of this development; its chromophore is both a blessing (to monitor the extent of native state formation) and a curse (since it complicates refolding kinetic studies). Reversible folding is also the holy grail for a-helical MPs; they are generally so hydrophobic (unlike OMPs) that they cannot be refolded from the urea- or guanidine-chloride denatured random coil state but have to be solubilized in SDS which induces an a-helical conformation that is more or less retained (though probably significantly rearranged) in the native state. A great advantage of using SDS as denaturant is that its denaturation potency can be systematically reduced by titrating in nonionic detergents such as dodecyl maltoside; although mixed micelles are undoubtedly not bona fide vesicles, this ‘‘tuning’’ of denaturation leads to fully reversible unfolding under equilibrium conditions and also opens up for comprehensive kinetic folding/ unfolding studies. So far we have protein engineering studies of the folding of three a-helical MPs in mixed SDS/DDM micelles, namely bacteriorhodopsin, the 4-TM DsbB and most recently the 6-TM membrane protease GlpG. These help to gradually build up
Editorial / Archives of Biochemistry and Biophysics 564 (2014) 262–264
a picture of a transition state which is closer to the SDS-denatured state than the native state in terms of compactness but clearly has defined regions of folded structure which are quite sharply segregated or even polarized. A novel observation is that GlpG appears to have a large region of ‘‘frustrated structure’’ surrounding a rather small folding nucleus while most of the C-terminal part is essentially unfolded; temptingly, these data highlight the N-terminus as the initiation of folding, just as we would expect in vivo. The next step is to connect this experimental insight with computational studies, which we expect will be the next great frontier in a-helical MP folding. Progressing from monomeric proteins, the final step is to understand how polytopic a-helical MPs associate to form complexes and oligomers. This work has been spearheaded by the trimeric diacyl glycerate kinase, the original model system for mixed micelle folding, where monomeric subunits fold (but not to completion) before trimerizing. The aquaglyceroporin GlpF is tetrameric in the native state, but can dissociate and unfold under mildly acidic conditions or in SDS; the latter case is completely reversible and proceeds through at least one moltenglobule-like dimer. In contrast, the potassium channel KcsA resists SDS and only dissociates and unfolds in trifluoroethanol, which is only completely reversible if small amounts of lipid is present. While the sugar transporter lacY is monomeric and unfolds reversibly to some extent in urea, another member of the major facilitator superfamily GalP is naturally trimeric, but can also be partially (and reversibly) unfolded in urea in the presence of DDM. This susceptibility to urea is probably due to the proteins’ low stability and high flexibility, which makes it easier to disrupt hydrophobic interactions holding the protein together. We summarize all these observations in 6 empirical rules, before moving on to a final section on how polytopic MPs oligomerize. Homo-oligomerization, often to form highly symmetric structures, is widespread among these MPs, probably caused by gene duplication. The degree of cooperativity between different monomeric units varies – in some cases only the oligomer is functional and is formed by cooperative folding while in other cases the monomers are the functional units. Cooperativity between monomers is best probed by genetic fusion of the protomers and specific introduction of mutations in one or more protomers. We categorize oligomerization in three classes; monomer folding and oligomerization may be tightly coupled (two-state or co-translational, typical for homo-oligomers such as channel proteins), they may constitute separate steps (three steps, most likely the case for hetero-oligomers) and finally oligomerization may be controlled by ligands or depend on cellular signals, as seen for G-protein coupled receptors. Oligomerization confers certain advantages: besides thermodynamic and biological (e.g. against proteases) stabilization of the protein, they provide an opportunity to evolve new functions, e.g. by modulating monomer–oligomer equilibria and opening up for allosteric regulation. This is summarized in 7 simple observational rules which point the way for future research in this area. After this guided tour of the mechanisms by which membrane proteins fold in membrane-like environments, the review by Heedeok Hong turns to the driving forces that make this process favorable – with a keen eye to the differences between water-soluble and membrane proteins [3]. The complexity of the membrane environment makes it clear that although the underlying physical principles of course are the same, the game rules determining MP stability differ fundamentally from those of water-soluble proteins. First of all, it might even be questioned whether folding is thermodynamically driven or under kinetic control. Reconstitution of bacteriorhodopsin from different denatured states and from fragments settled this in favor of thermodynamic control (at least in vitro) and led to Popot and Engelman’s seminal two-stage model for insertion and association of a-helical MPs. As discussed in the previous article, folding reversibility can be tested by mixed micelles and also
263
elaborated by combination with hydrogen–deuterium exchange, though it should be noted that the structure of the denatured state can vary with absolute SDS concentration. Therefore alternative approaches are needed: these include pulse-proteolysis and more recently steric trapping, in which unfolding allows binding of bulky tags like streptavidin; unlike conventional equilibrium denaturation (but like refolding kinetics), this allows direct measurement of stability at very low SDS concentrations and may help avoid incorrect linear extrapolations under these conditions – though data with GlpG folding kinetics indicate that linear correlations can exist at low SDS mole fractions. So what stabilizes these membrane proteins? The hydrophobic effect is a strong force in watersoluble protein folding, burying hydrophobic residues away from the hydrophilic solvent. However, this effect cannot be so pronounced in membrane proteins. Rather, efficient packing of buried residues, facilitated by small residues, optimizes (weak) van der Waals interactions in competition with lipid contacts – though there is apparently still room for improvement in many membrane proteins. Also, hydrogen bonding in MPs comes with a twist: close packing promotes CaHAO@C hydrogen bonds (particularly in dimerizing peptides), while more conventional hydrogen bonds seem to stabilize MPs to the same extent as water-soluble proteins. These effects may be modulated by penetrating water molecules, and changes in the hydrogen bonding pattern may even modulate overall helix architecture (kinked versus straight). Salt bridges between side chains with complementary charges are only weakly stabilizing in an aqueous environment while buried salt bridges do not stabilize water-soluble proteins more than their hydrophobic side chain replacements. There are no detailed studies of these interactions in a-helical MPs, and it may be difficult to extrapolate from data in OMPs due to major differences in polarity. Cation-p interactions, widespread in water-soluble proteins, go a step further in MPs where they mediate contact with the surrounding lipid and may rationalize the preponderance of aromatic residues in the interfacial region (which is particularly widespread in beta-barrel proteins). Cation–cation interactions only provide modest contributions to water-soluble protein stability, but their geometric specificity (favoring an edge-to-face configuration) likely contributes to specific interhelical contacts, though this remains to be explored in more detail – along with better modeling and mimicry of the specific membrane environment. Thus our understanding of forces specifically stabilizing MPs is probably only at the beginning of a steep learning curve which will be very exciting to follow. We should certainly not consider membrane protein studies to be an intractable problem. Jean-Luc Popot, whose two-stage model developed with Donald Engelman has done so much to stimulate thinking about membrane protein insertion and folding, is uniquely placed to prepare an overview of the current situation. He provides a veritable tour de force of the many different conditions used to fold and refold a-helical MPs and OMPs [4]. His compilation encompasses the 89 MPs (52 a-helical and 37 OMPs, of which 10 and 8, respectively, are oligomers) which had been reported to be folded in vitro by the end of 2013 (a number which far exceeded his initial expectations). The cases include both refolded (i.e. first denatured and then refolded) and folded (not previously native) proteins and span a number of different conditions. His article takes us on a historical survey of how folding experiments were important in developing our understanding of what a-helical MPs and OMPs ‘‘really are’’, how they behave in relation to the Anfinsen folding paradigm and the steps beyond simple dilution experiments (used for water-soluble proteins) to fold MPs. Interestingly, of the 42 MPs folded from inclusion bodies, around two thirds (27) are OMPs; the difficulty of folding a-helical MPs in this way may have stimulated the use of cell-free extracts for their expression, which is also a burgeoning field in membrane protein production. The table also lists more exotic denaturing and
264
Editorial / Archives of Biochemistry and Biophysics 564 (2014) 262–264
folding media such as sarkosyl and organic solvents and highlights helpers such as natural ligands as well as specific (non-annular) lipids. The actual mode of transfer from denaturing to folding conditions merits comments since it can be done in a variety of ways, though it generally appears that ‘‘faster is better’’. The great variety of ways to obtain folded MPs should not be seen as a bewildering obstacle but rather a versatile and helpful guide, which makes it more likely that you will eventually reach your goal and bring your own favorite MP to a safe (native) haven. As a final perspective on MP protein folding, Jean-Luc Popot continues with Jörg Kleinschmidt in a description of a synthetic group of amphipathic polymers called amphipols (APols) which hold great promise both in folding and stabilizing membrane proteins [5]. Like detergents, they contain both hydrophilic groups and hydrophobic chains, but as (short) polymers they interact less invasively with MPs than detergents do. Their polymeric nature means that they do not dissociate from MPs upon dilution (unless competed out by other detergents), which is a distinct advantage when handling MPs. Different APols are now available with different head-groups and thus different pH-sensitivities; recent additions remain water soluble even below pH 7 (the original APol, A8-35, has an ionizable carboxylate group and precipitates below pH 7). APols are superior to most conventional nonionic detergents (though not DDM) in resolubilizing bacteriorhodopsin and can replace SDS in a relatively straightforward manner. Bacteriorhodopsin is a robust protein, however. The real test is to see how APols perform with fragile G-protein coupled receptors and here APols are also promising; even without optimization, folding yields beyond 30% can be obtained for a variety of these receptors, heralding their potential in crystallization trials. Non-ionic APols (NAPols) also show promise, since their lack of charge reduces their unspecific binding to MPs and makes them superior to A8-35 in cell-free expression. APols are also good tools for folding of OMPs, since larger OMPs can be difficult to fold quantitatively; however, the 14-strand OMP FomA folds to 90% within 24 h with the expected gain of structure and functionality, while folding of OmpA into A8-35 shows a lower activation energy than folding into lipid bilayers. Experiments with new APols such as sulfonated
forms are also underway. Furthermore, once folded into APols, the MPs show much higher stability than in nonionic detergents, probably due to their tighter association as polymers. OmpA is less thermodynamically stable in A8-35 than in the nonionic detergent LDAO, but this may reflect the high pH of 10 required for folding, which will exacerbate electrostatic repulsion; nevertheless, it unfolds more slowly in A8-35 than in LDAO. These many intriguing observations can be explained as a combination of different phenomena: firstly, APols do not need to be present in excess (unlike detergents) and therefore do not constitute a large hydrophobic sink for e.g. monomerizing oligomeric MPs. However, even at concentrations comparable with detergents, APols stabilize MPs, since their polymeric structure and short hydrophobic chains makes it difficult for them to compete with protein/protein and protein/ lipid interactions. When they do bind MPs, they may do so by a ‘‘Gulliver effect’’, binding at multiple points and dampening or freezing MPs’ conformational fluctuations, as well as sterically preventing aggregation. These properties make APols a prominent member of the growing tool-box for membrane solubilization. I hope that you will enjoy reading these articles which highlight both problems and possibilities in this exciting field of research. I am very grateful to the many colleagues who have contributed to this and who have made it such a pleasure to edit this collection. References [1] L.M. McMorran, D.J. Brockwell, S.E. Radford, Arch. Biochem. Biophys. 564 (2014) 265–280. [2] J. Neumann, N. Klein, D.E. Otzen, D. Schneider, Arch. Biochem. Biophys. 564 (2014) 281–296. [3] H. Hong, Arch. Biochem. Biophys. 564 (2014) 297–313. [4] J.L. Popot, Arch. Biochem. Biophys. 564 (2014) 314–326. [5] J.H. Kleinschmidt, J.L. Popot, Arch. Biochem. Biophys. 564 (2014) 327–343.
Daniel Otzen Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Center for Insoluble Protein Structures (inSPIN), Aarhus University, Gustav Wieds Vej 14, DK – 8000 Aarhus C, Denmark Received 31 October 2014 Available online 1 November 2014
