Folding Of Soluble Proteins Research Articles

Helical reorganization in the context of membrane protein folding: Insights from simulations with bacteriorhodopsin (BR) fragments

Membrane protein folding is distinct from folding of soluble proteins. Conformational acquisition in major membrane protein subclasses can be delineated into insertion and folding processes. An exception to the “two stage” folding, later developed to “three stage” folding, is observed within the last two helices in bacteriorhodopsin (BR), a system that serves as a model membrane protein. We employ a reductionist approach to understand interplay of molecular factors underlying the apparent defiance. Leveraging available solution NMR structures, we construct, sample in silico, and analyze partially (PIn) and fully inserted (FIn) BR membrane states. The membrane lateral C-terminal helix (CH) in PIn is markedly prone to transient structural distortions over microsecond timescales; a disorder prone region (DPR) is thereby identified. While clear transmembrane propensities are not acquired, the distortions induce alterations in local membrane curvature and area per lipid. Importantly, energetic decompositions reveal that overall, the N-terminal helix (NH) is thermodynamically more stable in the PIn. Higher overall stability of the FIn arises from favorable interactions between the NH and the CH. Our results establish lack of spontaneous transition of the PIn to the FIn, and attributes their partitioning to barriers that exceed those accessible with thermal fluctuations. This work paves the way for further detailed studies aimed at determining the thermo-kinetic roles of the initial five helices, or complementary external factors, in complete helical folding and insertion in BR. We comment that complementing such efforts with the growing field of machine learning assisted energy landscape searches may offer unprecedented insights.

Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins.

Single-molecule tweezers, such as magnetic tweezers, are powerful tools for probing nm-scale structural changes in single membrane proteins under force. However, the weak molecular tethers used for the membrane protein studies have limited the observation of long-time, repetitive molecular transitions due to force-induced bond breakage. The prolonged observation of numerous transitions is critical in reliable characterizations of structural states, kinetics, and energy barrier properties. Here, we present a robust single-molecule tweezer method that uses dibenzocyclooctyne cycloaddition and traptavidin binding, enabling the estimation of the folding 'speed limit' of helical membrane proteins. This method is >100 times more stable than a conventional linkage system regarding the lifetime, allowing for the survival for ~12 hr at 50 pN and ~1000 pulling cycle experiments. By using this method, we were able to observe numerous structural transitions of a designer single-chained transmembrane homodimer for 9 hr at 12 pN and reveal its folding pathway including the hidden dynamics of helix-coil transitions. We characterized the energy barrier heights and folding times for the transitions using a model-independent deconvolution method and the hidden Markov modeling analysis, respectively. The Kramers rate framework yields a considerably low-speed limit of 21 ms for a helical hairpin formation in lipid bilayers, compared to μs scale for soluble protein folding. This large discrepancy is likely due to the highly viscous nature of lipid membranes, retarding the helix-helix interactions. Our results offer a more valid guideline for relating the kinetics and free energies of membrane protein folding.

Mechanisms of Protein Quality Control in the Endoplasmic Reticulum by a Coordinated Hsp40-Hsp70-Hsp90 System

The Hsp40, Hsp70, and Hsp90 chaperone families are ancient, highly conserved, and critical to cellular protein homeostasis. Hsp40 chaperones can transfer their protein clients to Hsp70, and Hsp70 can transfer clients to Hsp90, but the functional benefits of these transfers are unclear. Recent structural and mechanistic work has opened up the possibility of uncovering how Hsp40, Hsp70, and Hsp90 work together as unified system. In this review, we compile mechanistic data on the ER J-domain protein 3 (ERdj3) (an Hsp40), BiP (an Hsp70), and Grp94 (an Hsp90) chaperones within the endoplasmic reticulum; what is known about how these chaperones work together; and gaps in this understanding. Using calculations, we examine how client transfer could impact the solubilization of aggregates, the folding of soluble proteins, and the triage decisions by which proteins are targeted for degradation. The proposed roles of client transfer among Hsp40-Hsp70-Hsp90 chaperones are new hypotheses, and we discuss potential experimental tests of these ideas.

Impact of bacterial chaperonin GroEL\u2013GroES on bacteriorhodopsin folding and membrane integration

Our understanding of molecular chaperone function in membrane protein biogenesis lags far behind that in soluble protein biogenesis. Through a combined approach including isothermal titration calorimetry, UV–Vis spectroscopy, and fluorescence spectroscopy, the behavior of ATP-dependent chaperonin GroEL–GroES, a paradigmatic chaperone of soluble protein folding, was investigated in the refolding of membrane protein bacteriorhodopsin (BR) and its membrane insertion. We found that BR bound asymmetrically to the double-ring GroEL, with a much higher affinity when it was partially denatured. GroEL alone showed a clear influence on BR refolding, but the presence of ATP was necessary to significantly enhance both the rate and yield of the GroEL-mediated folding, in contrast to the adverse effect of GroES on the folding yield. However, synergy between ATP and GroES was shown to be required not only for releasing high-affinity BR species from GroEL, but also for unfolding and rescuing the misfolded conformers complexed to GroEL. This is consistent with the observation that maximum rate enhancement of BR refolding or assembly with the prepared inverted membrane vesicles was achieved when the complete chaperonin system was used. Our results support the iterative unfolding mechanism of GroEL activity previously proposed for soluble proteins, whereby GroEL might perform repeated unfolding and release of BR, thus offering additional opportunities for timely folding or membrane integration. This work provides important information on the convergence of folding of membrane and soluble proteins in light of folding pathways and the role of molecular chaperones.

Conformational dynamics of a membrane protein chaperone enables spatially regulated substrate capture and release

Membrane protein biogenesis poses enormous challenges to cellular protein homeostasis and requires effective molecular chaperones. Compared with chaperones that promote soluble protein folding, membrane protein chaperones require tight spatiotemporal coordination of their substrate binding and release cycles. Here we define the chaperone cycle for cpSRP43, which protects the largest family of membrane proteins, the light harvesting chlorophyll a/b-binding proteins (LHCPs), during their delivery. Biochemical and NMR analyses demonstrate that cpSRP43 samples three distinct conformations. The stromal factor cpSRP54 drives cpSRP43 to the active state, allowing it to tightly bind substrate in the aqueous compartment. Bidentate interactions with the Alb3 translocase drive cpSRP43 to a partially inactive state, triggering selective release of LHCP's transmembrane domains in a productive unloading complex at the membrane. Our work demonstrates how the intrinsic conformational dynamics of a chaperone enables spatially coordinated substrate capture and release, which may be general to other ATP-independent chaperone systems.

The Versatile Beta-Barrel Gives Up Secrets of the Membrane

The protein-folding problem is one of the contemporary challenges of biophysics. Knowing how the sequence encodes a native structure will provide insight into how genetic mutations cause disease, how protein sequences evolve and represents a tool to design proteins with novel structures and functions. While the folding of soluble proteins has been studied for decades, studies on membrane proteins are not as common. In this talk, I will present our recent developments in membrane protein stability measurements in vitro as well as their implications in living cells.

Site-Specific Information on Membrane Protein Folding by Electron Spin Echo Envelope Modulation Spectroscopy

Compared to folding of soluble proteins, folding of membrane proteins is complicated by the fact that it requires an amphiphilic environment. Few existing techniques can provide structurally resolved information on folding kinetics. For the major plant light harvesting complex LHCII, it is demonstrated that changes in water accessibility of a particular amino acid residue can be followed during folding by measuring the hyperfine interaction of spin labels with deuterium nuclei of heavy water. The incorporation of residue 196 into the hydrophobic core of a detergent micelle was investigated. The technique provides a time constant that is similar to the one found with fluorescence spectroscopy for the slower folding step of the whole protein and with electron paramagnetic resonance for change of the distance between residues 90 and 196. If applied to several residues, this technique should provide information on the sequence of events during membrane protein folding.

Studying Membrane Protein Thermodynamics Using a Steric Trap

An understanding of the molecular forces that specify a protein's structure is essential for many of the central quests of structural biology. While there has been a larger effort to understand soluble protein folding, we know very little about membrane protein folding energetics. We are developing a novel method to study the forces that stabilize membrane proteins in lipid bilayers, which we termed the ‘Steric Trap method.’ The steric trap method couples protein unfolding to a measurable binding event by exploiting steric repulsion and the high affinity of the streptavidin/biotin interaction. To do this, we introduce two biotin tags on a target protein that are close in space and employ monovalent streptavidin (mSA) as our steric trap. A single mSA can bind without steric hindrance to the folded protein, but a second mSA can only bind when the protein unfolds due to steric overlap. Thus, the binding affinity of the second streptavidin provides a measure of unfolding free energy because binding is coupled to unfolding. We have developed this method on the water-soluble protein, dihydrofolate reductase (DHFR). When two biotin-labeling sites were rationally designed to be close to one another in space and near the enzyme active site of DHFR, enzymatic activity was reversibly abolished upon incubation with a molar excess of monovalent streptavidin. Incubation with a stabilizing ligand shifted streptavidin binding curves, confirming that the steric trapping can quantitatively detect changes in protein stability. Results on our application of the steric trap method to membrane proteins will be presented. The steric trap method could be a powerful tool for measuring protein association affinities, studying unfolding energetics and investigating membrane protein unfolded states in the context of membrane environments.

Protein–protein interactions in carotenoid triggered quenching of phycobilisome fluorescence in Synechocystis sp. PCC 6803

An inquiry into the effect of temperature on carotenoid triggered quenching of phycobilisome (PBS) fluorescence in a photosystem II-deficient mutant of Synechocystis sp. results in identification of two temperature-dependent processes: one is responsible for the quenching rate, and one determines the yield of PBS fluorescence. Non-Arrhenius behavior of the light-on quenching rate suggests that carotenoid-absorbed light triggers a process that bears a strong resemblance to soluble protein folding, showing temperature-dependent enthalpy of activated complex formation. The response of PBS fluorescence yield to hydration changing additives and to passing of the membrane lipid phase transition point indicates that the pool size of PBSs subject to quenching depends on the state of some membrane component.

Refolding a beta-barrel membrane protein

The field of membrane protein folding is relatively new compared to soluble protein folding. This thesis describes spectroscopy investigations of the refolding and dynamics of a β-barrel membrane protein. The amphiphilic, β-barrel outer membrane protein A (OmpA) refolds and inserts directly into a lipid vesicle or micelle from a denatured state in aqueous urea solution. Spectroscopic probes used to study this system are native tryptophans located at positions 7, 15, 57, 102, and 143. Steady-state and time-resolved fluorescence measurements were performed using single tryptophan mutants of full-length OmpA (325 residues) and the truncated, transmembrane domain (176 residues). Both full-length and truncated mutants exhibit similar tryptophan emission lifetimes, suggesting that the transmembrane microenvironment is not greatly perturbed by the presence of the C-terminus. While the microenvironments of folded full-length and truncated OmpA appear similar, the dynamics of refolding at each tryptophan position exhibit subtle differences when the C-terminus is present. Specifically, we observe that tryptophan-102, which faces the pore interior, inserts and folds the fastest while tryptophan-7, which does not cross the bilayer, is the slowest. Fluorescence anisotropy decays also indicate that tryptophan-7 is the most flexible residue compared to the other tryptophans. Temperature studies below the lipid gel-liquid transition temperature have also been performed. In the lipid gel phase, OmpA adsorbs to the surface of the vesicles but contains immediate β-sheet structure upon folding as well as very hydrophobic tryptophan environments. It is still uncertain from ensemble measurements whether this species is a true intermediate. Fluorescence energy transfer kinetics have successfully determined the intramolecular distance between tryptophan-7 and cysteine-175 labeled with a dansyl fluorophore. These results reveal that the barrel ends of OmpA come into contact early in the refolding process and remain close together up to the final assembly of the barrel. We also have evidence that the adsorbed species at low temperatures is not an intermediate in the folding pathway since no energy transfer is observed for this species. These spectroscopic investigations have provided the foundation for further fundamental studies to dissect the molecular mechanism of the folding pathway of OmpA as well as other integral membrane proteins.

Lipid Solvation Effects Contribute to the Affinity of Gly-xxx-Gly Motif-Mediated Helix−Helix Interactions

Interactions between transmembrane helices are mediated by the concave Gly-xxx-Gly motif surface. Whether Gly residues per se are sufficient for selection of this motif has not been established. Here, we used the in vivo TOXCAT assay to measure the relative affinities of all 18 combinations of Gly, Ala, and Ser "small-xxx-small" mutations in glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP) homodimers. Affinity values were compared with the accessibility to a methylene-sized probe of the total surface area of each helix monomer as a measure of solvation by membrane components. A strong inverse correlation was found between nonpolar-group lipid accessibility and dimer affinity (R = 0.75 for GpA, p = 0.013, and R = 0.81 for MCP, p = 0.004), suggesting that lipid as a poor membrane protein solvent, conceptually analogous to water in soluble protein folding, can contribute to dimer stability and help to define helix-helix interfaces.

Proteins as nano-machines: dynamics–function relations studied by neutronscattering

A protein is a nano-machine whose molecular structure was selected by evolutionto perform specific biological functions. Neutron spectroscopy is uniquely suited toprovide experimental data on atomic motions in a protein under the influence offorces that maintain its stable and active molecular structure. Experiments arereviewed relating such a dynamics to soluble protein folding and stabilityin different environments, and to the activity of bacteriorhodopsin, amembrane protein nano-machine with light-driven proton pump activity.

4] Protein folding in membranes: Determining energetics of peptide-bilayer interactions

Although the problem of the folding of soluble proteins continues to resist solution, we at least have a strong understanding of the general thermodynamic principles1,2 and have available a wealth of thermodynamic data.3-5 The study of membrane protein folding and stability is much less advanced: Some general principles are emerging,6-9 but the amount of thermodynamic data available remains quite limited. The energetics of the partitioning of peptides into membranes constitutes one especially important class of data. We will demonstrate how such data can be used for clarifying the folding of peptides and small proteins in membranes and then describe the principles and methods used for determining the energetics of the partitioning of peptides into bilayer membranes.