Steric trapping strategy for studying the folding of helical membrane proteins.

Elucidating the folding energy landscape of membrane proteins is essential to the understanding of the proteins' stabilizing forces, folding mechanisms, biogenesis, and quality control. This is not a trivial task because the reversible control of folding is inherently difficult in a lipid bilayer environment. Recently, novel methods have been developed, each of which has a unique strength in investigating specific aspects of membrane protein folding. Among such methods, steric trapping is a versatile strategy allowing a reversible control of membrane protein folding with minimal perturbation of native protein-water and protein-lipid interactions. In a nutshell, steric trapping exploits the coupling of spontaneous denaturation of a doubly biotinylated protein to the simultaneous binding of bulky monovalent streptavidin molecules. This strategy has been evolved to investigate key elements of membrane protein folding such as thermodynamic stability, spontaneous denaturation rates, conformational features of the denatured states, and cooperativity of stabilizing interactions. In this review, we describe the critical methodological advancement, limitation, and outlook of the steric trapping strategy.

Lipid Bilayer Strengthens the Cooperative Network of a Membrane-Integral Enzyme.

Lipid bilayer provides a two-dimensional hydrophobic solvent milieu for membrane proteins in cells. Although the native bilayer is widely recognized as an optimal environment for folding and function of membrane proteins, the underlying physical basis remains elusive. Here, employing the intramembrane protease GlpG of Escherichia coli as a model, we elucidate how the bilayer stabilizes a membrane protein and engages the protein's residue interaction network compared to the nonnative hydrophobic medium, micelles. We find that the bilayer enhances GlpG stability by promoting residue burial in the protein interior compared to micelles. Strikingly, while the cooperative residue interactions cluster into multiple distinct regions in micelles, the whole packed regions of the protein act as a single cooperative unit in the bilayer. Molecular dynamics (MD) simulation indicates that lipids less efficiently solvate GlpG than detergents. Thus, the bilayerinduced enhancement of stability and cooperativity likely stems from the dominant intraprotein interactions outcompeting the weak lipid solvation. Our findings reveal a foundational mechanism in the folding, function, and quality control of membrane proteins. The enhanced cooperativity benefits function facilitating propagation of local structural perturbation across the membrane. However, the same phenomenon can render the proteins' conformational integrity vulnerable to missense mutations causing conformational diseases1,2.

Evolutionary balance between foldability and functionality of a glucose transporter.

Despite advances in resolving the structures of multi-pass membrane proteins, little is known about the native folding pathways of these complex structures. Using single-molecule magnetic tweezers, we here report a folding pathway of purified human glucose transporter 3 (GLUT3) reconstituted within synthetic lipid bilayers. The N-terminal major facilitator superfamily (MFS) fold strictly forms first, serving as a structural template for its C-terminal counterpart. We found polar residues comprising the conduit for glucose molecules present major folding challenges. The endoplasmic reticulum membrane protein complex facilitates insertion of these hydrophilic transmembrane helices, thrusting GLUT3's microstate sampling toward folded structures. Final assembly between the N- and C-terminal MFS folds depends on specific lipids that ease desolvation of the lipid shells surrounding the domain interfaces. Sequence analysis suggests that this asymmetric folding propensity across the N- and C-terminal MFS folds prevails for metazoan sugar porters, revealing evolutionary conflicts between foldability and functionality faced by many multi-pass membrane proteins.

Untangling the complexity of membrane protein folding.

Delineating the folding steps of helical-bundle membrane proteins has been a challenging task. Many questions remain unanswered, including the conformation and stability of the states populated during folding, the shape of the energy barriers between the states, and the role of lipids as a solvent in mediating the folding. Recently, theoretical frames have matured to a point that permits detailed dissection of the folding steps, and advances in experimental techniques at both single-molecule and ensemble levels enable selective modulation of specific steps for quantitative determination of the folding energy landscapes. We also discuss how lipid molecules would play an active role in shaping the folding energy landscape of membrane proteins, and how folding of multi-domain membrane proteins can be understood based on our current knowledge. We conclude this review by offering an outlook for emerging questions in the study of membrane protein folding.

Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein.

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Structural cavities are critical to balancing stability and activity of a membrane-integral enzyme.

Packing interaction is a critical driving force in the folding of helical membrane proteins. Despite the importance, packing defects (i.e., cavities including voids, pockets, and pores) are prevalent in membrane-integral enzymes, channels, transporters, and receptors, playing essential roles in function. Then, a question arises regarding how the two competing requirements, packing for stability vs. cavities for function, are reconciled in membrane protein structures. Here, using the intramembrane protease GlpG of Escherichiacoli as a model and cavity-filling mutation as a probe, we tested the impacts of native cavities on the thermodynamic stability and function of a membrane protein. We find several stabilizing mutations which induce substantial activity reduction without distorting the active site. Notably, these mutations are all mapped onto the regions of conformational flexibility and functional importance, indicating that the cavities facilitate functional movement of GlpG while compromising the stability. Experiment and molecular dynamics simulation suggest that the stabilization is induced by the coupling between enhanced protein packing and weakly unfavorable lipid desolvation, or solely by favorable lipid solvation on the cavities. Our result suggests that, stabilized by the relatively weak interactions with lipids, cavities are accommodated in membrane proteins without severe energetic cost, which, in turn, serve as a platform to fine-tune the balance between stability and flexibility for optimal activity.

Proteolysis mediated by the membrane-integrated ATP-dependent protease FtsH has a unique nonlinear dependence on ATP hydrolysis rates.

ATPases associated with diverse cellular activities (AAA+) proteases utilize ATP hydrolysis to actively unfold native or misfolded proteins and translocate them into a protease chamber for degradation. This basic mechanism yields diverse cellular consequences, including the removal of misfolded proteins, control of regulatory circuits, and remodeling of protein conformation. Among various bacterial AAA+ proteases, FtsH is only membrane-integrated and plays a key role in membrane protein quality control. Previously, we have shown that FtsH has substantial unfoldase activity for degrading membrane proteins overcoming a dual energetic burden of substrate unfolding and membrane dislocation. Here, we asked how efficiently FtsH utilizes ATP hydrolysis to degrade membrane proteins. To answer this question, we measured degradation rates of the model membrane substrate GlpG at various ATP hydrolysis rates in the lipid bilayers. We find that the dependence of degradation rates on ATP hydrolysis rates is highly nonlinear: (i) FtsH cannot degrade GlpG until it reaches a threshold ATP hydrolysis rate; (ii) after exceeding the threshold, the degradation rates steeply increase and saturate at the ATP hydrolysis rates far below the maxima. During the steep increase, FtsH efficiently utilizes ATP hydrolysis for degradation, consuming only 40-60% of the total ATP cost measured at the maximal ATP hydrolysis rates. This behavior does not fundamentally change against water-soluble substrates as well as upon addition of the macromolecular crowding agent Ficoll 70. The Hill analysis shows that the nonlinearity stems from coupling of three to five ATP hydrolysis events to degradation, which represents unique cooperativity compared to other AAA+ proteases including ClpXP, HslUV, Lon, and proteasomes.

The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network.

Intramembrane rhomboid proteases are of particular interest because of their function to hydrolyze a peptide bond of a substrate buried in the membrane. Crystal structures of the bacterial rhomboid protease GlpG have revealed a catalytic dyad (Ser201-His254) and oxyanion hole (His150/Asn154/the backbone amide of Ser201) surrounded by the protein matrix and contacting a narrow water channel. Although multiple crystal structures have been solved, the catalytic mechanism of GlpG is not completely understood. Because it is a serine protease, hydrogen bonding interactions between the active site residues are thought to play a critical role in the catalytic cycle. Here, we dissect the interaction energies among the active site residues His254, Ser201, and Asn154 of Escherichia coli GlpG, which form a hydrogen bonding network. We combine double mutant cycle analysis with stability measurements using steric trapping. In mild detergent, the active site residues are weakly coupled with interaction energies (ΔΔG Inter) of ‒1.4 kcal/mol between His254 and Ser201 and ‒0.2 kcal/mol between Ser201 and Asn154. Further, by analyzing the propagation of single mutations of the active site residues, we find that these residues are important not only for function but also for the folding cooperativity of GlpG. The weak interaction between Ser and His in the catalytic dyad may partly explain the unusually slow proteolysis by GlpG compared with other canonical serine proteases. Our result suggests that the weak hydrogen bonds in the active site are sufficient to carry out the proteolytic function of rhomboid proteases.

Native Proteomics in Discovery Mode Using Size-Exclusion Chromatography-Capillary Zone Electrophoresis-Tandem Mass Spectrometry.

Native proteomics aims to characterize complex proteomes under native conditions and ultimately produces a full picture of endogenous protein complexes in cells. It requires novel analytical platforms for high-resolution and liquid-phase separation of protein complexes prior to native mass spectrometry (MS) and MS/MS. In this work, size-exclusion chromatography (SEC)-capillary zone electrophoresis (CZE)-MS/MS was developed for native proteomics in discovery mode, resulting in the identification of 144 proteins, 672 proteoforms, and 23 protein complexes from the Escherichia coli proteome. The protein complexes include four protein homodimers, 16 protein-metal complexes, two protein-[2Fe-2S] complexes, and one protein-glutamine complex. Half of them have not been reported in the literature. This work represents the first example of online liquid-phase separation-MS/MS for the characterization of a complex proteome under the native condition, offering the proteomics community an efficient and simple platform for native proteomics.

Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH.

ATP-dependent protein degradation mediated by AAA+ proteases is one of the major cellular pathways for protein quality control and regulation of functional networks. While a majority of studies of protein degradation have focused on water-soluble proteins, it is not well understood how membrane proteins with abnormal conformation are selectively degraded. The knowledge gap stems from the lack of an in vitro system in which detailed molecular mechanisms can be studied as well as difficulties in studying membrane protein folding in lipid bilayers. To quantitatively define the folding-degradation relationship of membrane proteins, we reconstituted the degradation using the conserved membrane-integrated AAA+ protease FtsH as a model degradation machine and the stable helical-bundle membrane protein GlpG as a model substrate in the lipid bilayer environment. We demonstrate that FtsH possesses a substantial ability to actively unfold GlpG, and the degradation significantly depends on the stability and hydrophobicity near the degradation marker. We find that FtsH hydrolyzes 380-550 ATP molecules to degrade one copy of GlpG. Remarkably, FtsH overcomes the dual-energetic burden of substrate unfolding and membrane dislocation with the ATP cost comparable to that for water-soluble substrates by robust ClpAP/XP proteases. The physical principles elucidated in this study provide general insights into membrane protein degradation mediated by ATP-dependent proteolytic systems.

Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.

Membrane proteins are assembled through balanced interactions among proteins, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements of membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping, which couples the unfolding of a doubly biotinylated protein to the binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to the Escherichia coli intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.

Role of Lipids in Folding, Misfolding and Function of Integral Membrane Proteins.

The lipid bilayer that constitutes cell membranes imposes environmental constraints on the structure, folding and function of integral membrane proteins. The cell membrane is an enormously heterogeneous and dynamic system in its chemical composition and associated physical forces. The lipid compositions of cell membranes not only vary over the tree of life but also differ by subcellular compartments within the same organism. Even in the same subcellular compartment, the membrane composition shows strong temporal and spatial dependence on the environmental or biological cues. Hence, one may expect that the membrane protein conformations and their equilibria strongly depend on the physicochemical variables of the lipid bilayer. Contrary to this expectation, the structures of homologous membrane proteins belonging to the same family but from evolutionary distant organisms exhibit a striking similarity. Furthermore, the atomic structures of the same protein in different lipid environments are also very similar. This suggests that certain stable folds optimized for a specific function have been selected by evolution. On the other hand, there is growing evidence that, despite the overall stability of the protein folds, functions of certain membrane proteins require a particular lipid composition in the bulk bilayer or binding of specific lipid species. Here I discuss the specific and nonspecific modulation of folding, misfolding and function of membrane proteins by lipids and introduce several diseases that are caused by misfolding of membrane proteins.

Toward understanding driving forces in membrane protein folding.

α-Helical membrane proteins are largely composed of nonpolar residues that are embedded in the lipid bilayer. An enigma in the folding of membrane proteins is how a polypeptide chain can be condensed into the compact folded state in the environment where the hydrophobic effect cannot strongly drive molecular interactions. Probably other forces such as van der Waals packing, hydrogen bonding, and weakly polar interactions, which are regarded less important in the folding of water-soluble proteins, should emerge. However, it is not clearly understood how those individual forces operate and how they are balanced for stabilizing membrane proteins. Studying this problem is not a trivial task mainly because of the methodological challenges in controlling the reversible folding of membrane proteins in the lipid bilayer. Overcoming the hurdles, meaningful progress has been made in the field in the last few decades. This review will focus on recent studies tackling the problem of driving forces in membrane protein folding.

Measuring transmembrane helix interaction strengths in lipid bilayers using steric trapping.

We have developed a method to measure strong transmembrane (TM) helix interaction affinities in lipid bilayers that are difficult to measure by traditional dilution methods. The method, called steric trapping, couples dissociation of biotinylated TM helices to a competitive binding by monovalent streptavidin (mSA), so that dissociation is driven by the affinity of mSA for biotin and mSA concentration. By adjusting the binding affinity of mSA through mutation, the method can obtain dissociation constants of TM helix dimers (K d,dimer) over a range of six orders of magnitudes. The K d,dimer limit of measurable target interaction is extended 3-4 orders of magnitude lower than possible by dilution methods. Thus, steric trapping opens up new opportunities to study the folding and assembly of α-helical membrane proteins in lipid bilayer environments. Here we provide detailed methods for applying steric trapping to a TM helix dimer.

Membrane depth-dependent energetic contribution of the tryptophan side chain to the stability of integral membrane proteins.

Lipid solvation provides the primary driving force for the insertion and folding of integral membrane proteins. Although the structure of the lipid bilayer is often simplified as a central hydrophobic core sandwiched between two hydrophilic interfacial regions, the complexity of the liquid-crystalline bilayer structure and the gradient of water molecules across the bilayer fine-tune the energetic contributions of individual amino acid residues to the stability of membrane proteins at different depths of the bilayer. The tryptophan side chain is particularly interesting because despite its widely recognized role in anchoring membrane proteins in lipid bilayers, there is little consensus about its hydrophobicity among various experimentally determined hydrophobicity scales. Here we investigated how lipid-facing tryptophan residues located at different depths in the bilayer contribute to the stability of integral membrane proteins using outer membrane protein A (OmpA) as a model. We replaced all lipid-contacting residues of the first transmembrane ß-strand of OmpA with alanines and individually incorporated tryptophans in these positions along the strand. By measuring the thermodynamic stability of these proteins, we found that OmpA is slightly more stable when tryptophans are placed in the center of the bilayer and that it is somewhat destabilized as tryptophans approach the interfacial region. However, this trend may be partially reversed when a moderate concentration of urea rather than water is taken as the reference state. The measured stability profiles are driven by similar profiles of the m-value, a parameter that reflects the shielding of hydrophobic surface area from water. Our results indicate that knowledge of the free energy level of the protein's unfolded reference state is important for quantitatively assessing the stability of membrane proteins, which may explain differences in observed profiles between in vivo and in vitro scales.

Dramatic destabilization of transmembrane helix interactions by features of natural membrane environments.

Membrane proteins have evolved to fold and function in a lipid bilayer, so it is generally assumed that their stability should be optimized in a natural membrane environment. Yet optimal stability is not always in accord with optimization of function, so evolutionary pressure, occurring in a complex membrane environment, may favor marginal stability. Here, we find that the transmembrane helix dimer, glycophorin A (GpATM), is actually much less stable in the heterogeneous environment of a natural membrane than it is in model membranes and even common detergents. The primary destabilizing factors are electrostatic interactions between charged lipids and charged GpATM side chains, and nonspecific competition from other membrane proteins. These effects overwhelm stabilizing contributions from lateral packing pressure and excluded volume. Our work illustrates how evolution can employ membrane composition to modulate protein stability.

Method to measure strong protein-protein interactions in lipid bilayers using a steric trap.

Measuring high affinity protein-protein interactions in membranes is extremely challenging because there are limitations to how far the interacting components can be diluted in bilayers. Here we show that a steric trap can be employed for stable membrane interactions. We couple dissociation to a competitive binding event so that dissociation can be driven by increasing the affinity or concentration of the competitor. The steric trap design used here links monovalent streptavidin binding to dissociation of biotinylated partners. Application of the steric trap method to the well-characterized glycophorin A transmembrane helix (GpATM) reveals a dimer that is dramatically stabilized by 4-5 kcal/mol in palmitoyloleoylphosphatidylcholine bilayers compared to detergent. We also find larger effects of mutations at the dimer interface in bilayers compared to detergent suggesting that the dimer is more organized in a membrane environment. The high affinity we measure for GpATM in bilayers indicates that a membrane vesicle many orders of magnitude larger than a bacterial cell would be required to measure the dissociation constant using traditional dilution methods. Thus, steric trapping can open new biological systems to experimental scrutiny in natural bilayer environments.

Protein unfolding with a steric trap.

The study of protein folding requires a method to drive unfolding, which is typically accomplished by altering solution conditions to favor the denatured state. This has the undesirable consequence that the molecular forces responsible for configuring the polypeptide chain are also changed. It would therefore be useful to develop methods that can drive unfolding without the need for destabilizing solvent conditions. Here we introduce a new method to accomplish this goal, which we call steric trapping. In the steric trap method, the target protein is labeled with two biotin tags placed close in space so that both biotin tags can only be bound by streptavidin when the protein unfolds. Thus, binding of the second streptavidin is energetically coupled to unfolding of the target protein. Testing the method on a model protein, dihydrofolate reductase (DHFR), we find that streptavidin binding can drive unfolding and that the apparent binding affinity reports on changes in DHFR stability. Finally, by employing the slow off-rate of wild-type streptavidin, we find that DHFR can be locked in the unfolded state. The steric trap method provides a simple method for studying aspects of protein folding and stability in native solvent conditions, could be used to specifically unfold selected domains, and could be applicable to membrane proteins.

Methods for measuring the thermodynamic stability of membrane proteins.

Learning how amino acid sequences define protein structure has been a major challenge for molecular biology since the first protein structures were determined in the 1960s. In contrast to the staggering progress with soluble proteins, investigations of membrane protein folding have long been hampered by the lack of high-resolution structures and the technical challenges associated with studying the folding process in vitro. In the past decade, however, there has been an explosion of new membrane protein structures and a slower but notable increase in efforts to study the factors that define these structures. Here we review the methods that have been used to evaluate the thermodynamic stability of membrane proteins and provide some salient examples of how the methods have been used to begin to understand the energetics of membrane protein folding.

Role of aromatic side chains in the folding and thermodynamic stability of integral membrane proteins.

Aromatic residues are frequently found in helical and beta-barrel integral membrane proteins enriched at the membrane-water interface. Although the importance of these residues in membrane protein folding has been rationalized by thermodynamic partition measurements using peptide model systems, their contribution to the stability of bona fide membrane proteins has never been demonstrated. Here, we have investigated the contribution of interfacial aromatic residues to the thermodynamic stability of the beta-barrel outer membrane protein OmpA from Escherichia coli in lipid bilayers by performing extensive mutagenesis and equilibrium folding experiments. Isolated interfacial tryptophanes contribute -2.0 kcal/mol, isolated interfacial tyrosines contribute -2.6 kcal/mol, and isolated interfacial phenylalanines contribute -1.0 kcal/mol to the stability of this protein. These values agree well with the prediction from the Wimley-White interfacial hydrophobicity scale, except for tyrosine residues, which contribute more than has been expected from the peptide models. Double mutant cycle analysis reveals that interactions between aromatic side chains become significant when their centroids are separated by less than 6 A but are nearly insignificant above 7 A. Aromatic-aromatic side chain interactions are on the order of -1.0 to -1.4 kcal/mol and do not appear to depend on the type of aromatic residue. These results suggest that the clustering of aromatic side chains at membrane interfaces provides an additional heretofore not yet recognized driving force for the folding and stability of integral membrane proteins.