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• W4310592040 abstract "•The Hsp90 chaperone can actively compact its client protein chains•This induced compaction depends on ATP and performs mechanical work•The Hsp90 interactions can suppress the formation of misfolded and aggregated structures The chaperone heat shock protein 90 (Hsp90) is well known to undergo important conformational changes, which depend on nucleotide and substrate interactions. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally yet is central to its function. Here, using optical tweezers, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded, and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable clients and cooperates with Hsp70. The chaperone heat shock protein 90 (Hsp90) is well known to undergo important conformational changes, which depend on nucleotide and substrate interactions. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally yet is central to its function. Here, using optical tweezers, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded, and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable clients and cooperates with Hsp70. IntroductionThe heat shock protein 90 (Hsp90) chaperone is highly conserved and essential in eukaryotic cells. It is involved in many cellular functions, ranging from protection against heat stress to signal transduction and protein trafficking (Taipale et al., 2010Taipale M. Jarosz D.F. Lindquist S. HSP90 at the hub of protein homeostasis: emerging mechanistic insights.Nat. Rev. Mol. Cell Bio. 2010; 11: 515-528Crossref PubMed Scopus (0) Google Scholar), which often involve cooperation with Hsp70 (Genest et al., 2011Genest O. Hoskins J.R. Camberg J.L. Doyle S.M. Wickner S. Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling.Prod. Natl. Acad. Sci. USA. 2011; 108: 8206-8211Crossref PubMed Scopus (0) Google Scholar; Kirschke et al., 2014Kirschke E. Goswami D. Southworth D. Griffin P.R. Agard D.A. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.Cell. 2014; 157: 1685-1697Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Hsp90 interacts with many co-chaperones in eukaryotes (Echeverria et al., 2011Echeverria P.C. Bernthaler A. Dupuis P. Mayer B. Picard D. An interaction network predicted from public data as a discovery tool: application to the Hsp90 molecular chaperone machine.PLoS One. 2011; 6Crossref Scopus (156) Google Scholar; Mayer and Le Breton, 2015Mayer M.P. Le Breton L. Hsp90: breaking the symmetry.Mol. Cell. 2015; 58: 8-20Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), whereas no co-chaperone has been identified for the Escherichia coli Hsp90, also termed HtpG. Hsp90 undergoes large conformational changes during its ATPase cycle (Figure 1A ) (Krukenberg et al., 2009Krukenberg K.A. Southworth D.R. Street T.O. Agard D.A. pH-dependent conformational changes in bacterial Hsp90 reveal a grp94-like conformation at pH 6 that is highly active in suppression of citrate synthase aggregation.J. Mol. Biol. 2009; 390: 278-291Crossref PubMed Scopus (45) Google Scholar; Shiau et al., 2006Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Structural analysis of E-coli hsp90 reveals dramatic nucleotide-dependent conformational rearrangements.Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar; Street et al., 2011Street T.O. Lavery L.A. Agard D.A. Substrate binding drives large-scale conformational changes in the Hsp90 molecular chaperone.Mol. Cell. 2011; 42: 96-105Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Hsp90 interacts preferentially with unstable disordered regions (Schneider et al., 1996Schneider C. Sepp-Lorenzino L. Nimmesgern E. Ouerfelli O. Danishefsky S. Rosen N. Hartl F.U. Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90.Proc. Natl. Acad. Sci. USA. 1996; 93: 14536-14541Crossref PubMed Scopus (369) Google Scholar). The intrinsically disordered Tau bound an open state of Hsp90 in an ATP-independent manner, whereas ATP did affect the Hsp90 state of pre-formed Tau-Hsp90 complexes (Karagoz et al., 2014Karagoz G.E. Duarte A.M.S. Akoury E. Ippel H. Biernat J. Luengo T.M. Radli M. Didenko T. Nordhues B.A. Veprintsev D.B. et al.Hsp90-Tau complex reveals molecular basis for specificity in chaperone action.Cell. 2014; 156: 963-974Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The disordered ribosomal protein L2 accelerated ATP hydrolysis by Hsp90, suggesting it stimulates Hsp90 closure (Motojima-Miyazaki et al., 2010Motojima-Miyazaki Y. Yoshida M. Motojima F. Ribosomal protein L2 associates with E. coli HtpG and activates its ATPase activity.Biochem Bioph Res. Co. 2010; 400: 241-245Crossref PubMed Scopus (0) Google Scholar). HtpG bound a locally structured region of the partially folded Δ131Δ (Street et al., 2014Street T.O. Zeng X.H. Pellarin R. Bonomi M. Sali A. Kelly M.J.S. Chu F.X. Agard D.A. Elucidating the mechanism of substrate recognition by the bacterial Hsp90 molecular chaperone.J. Mol. Biol. 2014; 426: 2393-2404Crossref PubMed Scopus (37) Google Scholar), while Hsp90 bound the glucocorticoid receptor in open and closed states (Lorenz et al., 2014Lorenz O.R. Freiburger L. Rutz D.A. Krause M. Zierer B.K. Alvira S. Cuellar J. Valpuesta J.M. Madl T. Sattler M. et al.Modulation of the Hsp90 chaperone cycle by a stringent client protein.Mol. Cell. 2014; 53: 941-953Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In contrast to Δ131Δ and L2, however, binding here slowed Hsp90 closure and decelerated ATP hydrolysis. Overall, these findings indicate that substrate binding affects and depends on Hsp90 conformation.In contrast, it remains poorly understood how Hsp90 affects substrate conformations. Bacterial Hsp90 requires cooperation with Hsp70 to aid refolding (Genest et al., 2011Genest O. Hoskins J.R. Camberg J.L. Doyle S.M. Wickner S. Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling.Prod. Natl. Acad. Sci. USA. 2011; 108: 8206-8211Crossref PubMed Scopus (0) Google Scholar; Moran Luengo et al., 2018Moran Luengo T. Kityk R. Mayer M.P. Rudiger S.G.D. Hsp90 breaks the deadlock of the Hsp70 chaperone system.Mol. Cell. 2018; 70: 545-552.e549Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Hsp90 can reactivate luciferase with Hop/Sti1 (Johnson et al., 1998Johnson B.D. Schumacher R.J. Ross E.D. Toft D.O. Hop modulates hsp70/hsp90 interactions in protein folding.J. Biol. Chem. 1998; 273: 3679-3686Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar) and modulate the activity of steroid receptors with Hsp70 and co-chaperones (Morishima et al., 2000Morishima Y. Murphy P.J.M. Li D.P. Sanchez E.R. Pratt W.B. Stepwise assembly of a glucocorticoid receptor center dot hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket.J. Biol. Chem. 2000; 275: 18054-18060Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Hsp90 can facilitate de novo folding in the cell, where various additional factors are present (Thomas and Baneyx, 2000Thomas J.G. Baneyx F. ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells.Mol. Microbiol. 2000; 36: 1360-1370Crossref PubMed Scopus (109) Google Scholar). How unfolded protein chains are affected by Hsp90 (or HtpG) alone is incompletely resolved (Street et al., 2014Street T.O. Zeng X.H. Pellarin R. Bonomi M. Sali A. Kelly M.J.S. Chu F.X. Agard D.A. Elucidating the mechanism of substrate recognition by the bacterial Hsp90 molecular chaperone.J. Mol. Biol. 2014; 426: 2393-2404Crossref PubMed Scopus (37) Google Scholar). Addressing this issue is technically challenging. The disordered nature of Hsp90 substrates suggests that during their interaction with Hsp90, they may be characterized by conformational ensembles rather than distinct conformational states. Moreover, the diverse possible binding sites on Hsp90 indicate the possibility of additional conformational heterogeneity and dynamics that are difficult to characterize.Here, we study this issue by manipulating individual Firefly luciferase molecules, as well as maltose-binding protein (MBP) and the glucocorticoid receptor, using optical tweezers. We aim to study how interactions with bacterial Hsp90 (HtpG) locally affect the conformation of unfolded substrates rather than how Hsp90 assists in folding, which involves Hsp70 and other co-factors. Local structures that form in unfolded chains and hence change the distance between the N and C termini can be followed using optical tweezers, even if transient and heterogeneous. Our experiments showed HtpG can produce a gradual compaction in unfolded luciferase, and sudden stepwise contractions, in an ATP-stimulated manner. HtpG was found to suppress entry into a kinetically trapped state (luciferase) and aggregation between proteins (MBP). The data indicate that HtpG promotes local intra-chain contacts within client protein chains while suppressing distant contacts and induces a spectrum of transiently stable local conformations and overall chain compaction. This function may also have important implications for the interplay between Hsp90 and Hsp70 (Kirschke et al., 2014Kirschke E. Goswami D. Southworth D. Griffin P.R. Agard D.A. Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.Cell. 2014; 157: 1685-1697Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar; Rodriguez et al., 2008Rodriguez F. Arsene-Ploetze F. Rist W. Rudiger S. Schneider-Mergener J. Mayer M.P. Bukau B. Molecular basis for regulation of the heat shock transcription factor sigma32 by the DnaK and DnaJ chaperones.Mol. Cell. 2008; 32: 347-358Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar; Sharma et al., 2010Sharma S.K. De los Rios P. Christen P. Lustig A. Goloubinoff P. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase.Nat. Chem. Biol. 2010; 6: 914-920Crossref PubMed Scopus (158) Google Scholar). More generally, protein chain compaction or collapse is key to protein states including phase separation (Dill, 1985Dill K.A. Theory for the folding and stability of globular proteins.Biochemistry. 1985; 24: 1501-1509Crossref PubMed Google Scholar; Kim and Baldwin, 1982Kim P.S. Baldwin R.L. Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding.Annu. Rev. Biochem. 1982; 51: 459-489Crossref PubMed Google Scholar) and may be broadly relevant to protein quality control.ResultsStabilization of the unfolded state by Hsp90We surmised that one effect of Hsp90 could be the stabilization of unfolded substrate states, as the ability of Hsp90 to bind unfolded regions can compete with tertiary structure formation in the polypeptide chain. To investigate such stabilization of unfolded states by bacterial Hsp90 (HtpG) at the single-molecule level, we tethered a luciferase protein (Figure 1B) between two polystyrene beads via a DNA linker and unfolded it fully by mechanical stretching (Figure 1C). In these experiments, we continuously measure the distance between the beads, also referred to as the extension, as well as the force within the protein-DNA tether. Next, we relaxed the unfolded chain, waited at 0 pN for 5 s, and then stretched to assess whether it had remained unfolded or had formed tertiary structure (Mashaghi et al., 2014Mashaghi A. Mashaghi S. Tans S.J. Misfolding of luciferase at the single-molecule level.Angew Chem Int Edit. 2014; 53: 10390-10393Crossref PubMed Scopus (0) Google Scholar). For unfolded chains, stretching traces follow approximately a force-extension curve for a non-interacting polypeptide chain (of the length of luciferase) tethered to DNA, which is well predicted by the worm-like chain (WLC) model (Figure 1D, light blue traces) (Marko and Siggia, 1995Marko J.F. Siggia E.D. Stretching DNA.Macromolecules. 1995; 28: 8759-8770Crossref Google Scholar). For chains with tertiary structure, the stretching data initially display compact conformations, which subsequently show discrete unfolding transitions (Figure 1D, dark blue traces). In the absence of HtpG, we found that in 9% of the relaxation-stretching cycles, the chain remained in or near the unfolded state (Figure 1E, light blue bar). In the presence of HtpG and ATP, the first stretching curves for newly tethered luciferase proteins were similar to the curves without HtpG, indicating a lack of interaction. After full unfolding, we similarly continued with cycles of relaxation, waiting at 0 pN for 5 s, and stretching. Now, a larger fraction (24%, p < 0.05, Figure 1E, light blue bar) of the cycles showed that the chain remained in or near the unfolded state. These observations suggested that HtpG bound the unfolded chains, which is consistent with previous reports (Karagoz et al., 2014Karagoz G.E. Duarte A.M.S. Akoury E. Ippel H. Biernat J. Luengo T.M. Radli M. Didenko T. Nordhues B.A. Veprintsev D.B. et al.Hsp90-Tau complex reveals molecular basis for specificity in chaperone action.Cell. 2014; 156: 963-974Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar; Street et al., 2011Street T.O. Lavery L.A. Agard D.A. Substrate binding drives large-scale conformational changes in the Hsp90 molecular chaperone.Mol. Cell. 2011; 42: 96-105Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), and can hence stabilize the unfolded state.Protein chain compaction by Hsp90The relaxation data showed another type of effect on the unfolded luciferase substrates. Theory indicates that during relaxation to low force, an extended non-interacting protein chain coils up as described by the WLC force-extension curve (Figure 2A , blue curve). Unfolded luciferase chains without HtpG present typically displayed that behavior while showing deviations at lower forces (Figure 2B, below 5 pN). In presence of HtpG and ATP, relaxation data deviated more strongly, with chains compacting when the force decreased below 25 pN, as indicated by extensions smaller than the unfolded-chain WLC model. We observed chains that became similarly compact as fully folded states for forces below a few pN, with force-extension data following the WLC model for the folded state (Figures 2C and 2D). The compaction progressed gradually with decreasing force while displaying small stepwise contractions down to the resolution limit of about 10 nm (Figure 2D). Overall, these data indicated a gradual compaction of the protein chain.Figure 2HtpG compacts unfolded luciferase chainsShow full caption(A) Cartoons of a non-interacting chain that coils up when the force is decreased from 1 to 3, and corresponding force-extension curve.(B) Measured force-extension data for unfolded luciferase that is relaxed from high to low force, from different relax-stretch cycles and different molecules.(C) Cartoons of a chain that collapses to a compact state when the force is decreased from 1 to 3, and corresponding force-extension curve.(D) Measured force-extension data for unfolded luciferase that is relaxed from high to low force in presence of HtpG and ATP, from different relax-stretch cycles and different molecules.(E) Histogram of observed relaxation energies in the absence (light blue, n = 63 traces) and presence of HtpG and ATP (dark blue, n = 125 traces), as quantified by the surface area in between the relaxation trace and the theoretical worm-like chain model (top).(F) Measured stretching data from low to high force in presence of HtpG and ATP, from different relax-stretch cycles and different molecules.(G) Histogram of stretching energy, in the absence (light blue, n = 63 traces) and presence of HtpG and ATP (dark blue, n = 125 traces) as quantified by the surface area in between the stretching trace and the theoretical worm-like chain model (top).(H) Hysteresis quantification. Relaxation energy against the stretching energy of the subsequent stretching trace. Distance from the diagonal quantifies the hysteresis of the cycle. Points on the diagonal reflect a lack of hysteresis.See also Figure S1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To quantify the compaction effects, we determined the surface area under the relaxation traces (Figure 2E). We are interested in deviations from a non-interacting chain, and hence we subtracted the area corresponding WLC behavior, as well as that of the DNA linker (STAR Methods). Note that this surface area reflects mechanical work. Here, we refer to it as the “relaxation energy,” but note that it does not signify an equilibrium (free) energy. For the relaxation traces of luciferase without HtpG, this analysis showed a small, but non-zero, relaxation energy that extended up to 250 kBT (Figure 2E, light blue). The order of magnitude of these effects are consistent with previous atomic force microscopy pulling studies of the hydrophobic collapse of isolated protein chains (Walther et al., 2007Walther K.A. Grater F. Dougan L. Badilla C.L. Berne B.J. Fernandez J.M. Signatures of hydrophobic collapse in extended proteins captured with force spectroscopy.Prod. Natl. Acad. Sci. USA. 2007; 104: 7916-7921Crossref PubMed Scopus (0) Google Scholar). In line with the observed compaction, the distribution of the relaxation energy increased with HtpG, and ATP now extended to 600 kBT (Figure 2E, dark blue). Thus, HtpG induced contractions in the protein chain against a counter-acting force.Next, we tested whether human Hsp90 (hHsp90) also induced chain compactions in one of its clients, the glucocorticoid receptor ligand-binding domain (GRLBD). We found relaxation energies that were predominantly below 15 kBT in the absence of hHsp90 (Figure S1). In the presence of hHsp90 and ATP, the relaxation energies were predominantly above 15 kBT, and the histogram now displayed a shoulder extending up to 75 kBT (Figure S1). Thus, hHsp90 promoted a contraction in the GR chain despite the counter-acting forces acting within it. Overall, these data indicated that both bacterial and hHsp90 can compact protein chains.Hsp90 lowers the kinetic barrier to compacted statesIn addition to the area under the relaxation trace, which estimates the compaction energy, one may also quantify the area under the stretching traces (Figure 2F). For luciferase, this stretching energy was found to decrease on average in the presence of HtpG (Figure 2G; p < 0.05). Thus, whereas HtpG increased the relaxation energy, it decreased the stretching energy. These opposing trends indicated that the resulting compacted chain conformations, which are possibly complexed with HtpG, are less resistant to force than the folded conformations in the absence of HtpG. The gradual compaction was also found to display small local steps, which likely involve few residues that are close together along the protein chain. In contrast, larger global folding transitions often involve many residues that may be far apart along the protein chain.The notion of hysteresis can help to further discuss and explain this point. In general, hysteresis informs on the history dependence of a system. A system is non-hysteretic when its current state does not depend on its previous state. For example, for a theoretical non-interacting chain undergoing relaxation-stretching cycles, the measured extension depends on the currently applied force as given by the WLC model but not on previously applied forces. In such cases, the relaxation and stretching curves overlap, and the relaxation and stretching energies are identical. A different situation arises when considering (un)folding, for instance. As the tension is increased to a certain force, a chain that was folded may remain folded, but when the tension is decreased to that same force, the chain that was unfolded may remain unfolded. The delay in (un)folding is relevant here: both processes take time, which preserves previous states. The system is then not in thermodynamic equilibrium, and the difference in relaxation and stretching energies indicates the hysteresis. A limited hysteresis typically indicates low kinetic folding barriers and hence fast folding, as observed for small folds that exhibit limited cooperativity between residue-residue contacts (Jagannathan and Marqusee, 2013Jagannathan B. Marqusee S. Protein folding and unfolding under force.Biopolymers. 2013; 99: 860-869Crossref PubMed Scopus (42) Google Scholar). Conversely, larger tertiary structures with complex folds and many cooperative contacts tend to exhibit large kinetic barriers, slow folding, and significant hysteresis (Shank et al., 2010Shank E.A. Cecconi C. Dill J.W. Marqusee S. Bustamante C. The folding cooperativity of a protein is controlled by its chain topology.Nature. 2010; 465: 637-U134Crossref PubMed Scopus (183) Google Scholar).Here, we determine the hysteresis by the difference between the relaxation and stretching energies of each relax-stretch cycle, as assessed by plotting one against the other (Figure 2H). The distance from the diagonal then quantifies the hysteresis. In the absence of HtpG, the cycles typically were separated from the diagonal by a distance of order 100 kBT, which indicated substantial hysteresis, and is expected for a chain that folds (Figure 2H, light blue points). In the presence of HtpG and ATP, some cycles also displayed hysteresis, but many points were now close or on the diagonal, indicating limited to no hysteresis (Figure 2H, dark blue points). The relaxation (and stretching) energies were up to 400 kBT in these non-hysteretic cycles. Thus, while the resulting states were compacted like folded states, they displayed a lack of hysteresis. Such reversibility has been observed in the formation and disruption of collapsed states in individual hydrophobic polymers (Li and Walker, 2011Li I.T.S. Walker G.C. Signature of hydrophobic hydration in a single polymer.Prod. Natl. Acad. Sci. USA. 2011; 108: 16527-16532Crossref PubMed Scopus (0) Google Scholar). Note that the relaxation process takes 10 s and shows limited compaction without HtpG, while significant compaction occurs in the 5 s folding window. These observations and associated hysteresis are consistent with luciferase folding being efficiently suppressed by the applied force, which is present during relaxation but not during the folding time window. Conversely, the limited hysteresis in the presence of HtpG is consistent with the compaction steps being smaller; their formation can take place at higher applied force, while their disruption occurs at lower forces (compared with the no-chaperone case). Taken together, the decrease of hysteresis in the presence of HtpG suggests that Hsp90 lowers the kinetic barrier to compacted states.Suppression of misfolding by HtpGThe data so far indicated that HtpG can promote the formation of compacted structures of dimensions down to a similar order as fully folded states, within the measurement limits of our experiments. This compaction was gradual and displayed small steps (Figure 2D). Consistent with the reversibility observed in the previous section, the stretching traces also displayed similar gradual and small step features Figure 2F) and were hence distinct from the stretching traces in the absence of HtpG (Figure 1D, dark blue traces). Note that some stretching traces also showed the larger steps that were common in the absence of HtpG (Figures 2D and 1D), which may indicate that the luciferase polypeptides are not affected everywhere by HtpG and hence allows partial refolding of luciferase. These data are also consistent with previous bulk data showing that HtpG alone does not refold luciferase and rather requires cooperation with DnaK (Moran Luengo et al., 2018Moran Luengo T. Kityk R. Mayer M.P. Rudiger S.G.D. Hsp90 breaks the deadlock of the Hsp70 chaperone system.Mol. Cell. 2018; 70: 545-552.e549Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).A related issue is if Hsp90 affects entry into kinetically trapped misfolded states along the folding pathway. Luciferase is known to adopt non-native states by repeated freeze-thaw cycles (Genest et al., 2011Genest O. Hoskins J.R. Camberg J.L. Doyle S.M. Wickner S. Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling.Prod. Natl. Acad. Sci. USA. 2011; 108: 8206-8211Crossref PubMed Scopus (0) Google Scholar; Johnson et al., 1998Johnson B.D. Schumacher R.J. Ross E.D. Toft D.O. Hop modulates hsp70/hsp90 interactions in protein folding.J. Biol. Chem. 1998; 273: 3679-3686Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Luciferase has also been shown to adopt kinetically trapped states in mechanical relaxation-stretching cycles (Mashaghi et al., 2014Mashaghi A. Mashaghi S. Tans S.J. Misfolding of luciferase at the single-molecule level.Angew Chem Int Edit. 2014; 53: 10390-10393Crossref PubMed Scopus (0) Google Scholar). During the 5 s waiting time at 0 pN in stretching and relaxation cycles, and in the absence of chaperone, luciferase indeed formed compact structures, termed X states, that are characterized by a protein length (the length of the unstructured part of the protein) of about 105 nm (13% of the cycles; Figures 3A and 3B , red state). These states can be distinguished from larger (I1) and smaller (I2) intermediate structures (Figures 3A and 3B). These X states have been identified as kinetically trapped misfolded states as they have a significantly longer lifetime (minutes) and a maximally sustained force (43 pN on average) that is larger than other states of intermediate size (seconds and 24 pN, respectively) (Mashaghi et al., 2014Mashaghi A. Mashaghi S. Tans S.J. Misfolding of luciferase at the single-molecule level.Angew Chem Int Edit. 2014; 53: 10390-10393Crossref PubMed Scopus (0) Google Scholar). This maximally sustained force equals the unfolding force when unfolding occurs or the largest measured force when unfolding does not occur during stretching. In the presence of HtpG and ATP, the formation of misfolded X-state structures was almost abolished: their frequency was reduced more than 10-fold (from 13% to 0.8% of the cycles, p < 0.05; Figure 3C). The other folded states, including I1 and I2, did not show such a significant decrease (Figure 3C). Overall, the data indicated that HtpG suppressed luciferase misfolding.Figure 3Misfold suppression by HtpGShow full caption(A) Schematic representation of observed Firefly luciferase states ordered by protein length, ranging from fully compact (F; black) to fully unfolded (U; white) state (top panel). Also indicated are a misfolded state (X; red), and states that are more compact (I1; light gray) or less compact (I2; dark gray). Bottom panel: transitions between states observed in (B) during stretching (top arrows) and in relaxed states (bottom arrows).(B) Stretching and relaxation (blue traces), waiting at 0 pN for 5s, and stretching (red trace). Data show entry into state (X, red) of about 105 nm, which is stable over minutes and resists high forces, and corresponds to a misfolded state (Mashaghi et al., 2014Mashaghi A. Mashaghi S. Tans S.J. Misfolding of luciferase at the single-molecule level.Angew Chem Int Edit. 2014; 53: 10390-10393Crossref PubMed Scopus (0) Google Scholar). Gray lines are worm-like chain theoretical predictions of F and U states.(C) Corresponding fraction of observed entry into the different states indicated in (A), in the absence (light blue, n = 53 cycles) and presence (dark blue, n = 126 cycles) of bacterial Hsp90 (HtpG, 1μM). Cycles consist of relaxing chains in the fully unfolded state (right-most white circle), followed by a 5 s waiting time at 0 pN that provides an opportunity to adopt a different folded state. The latter is quantified in subsequent stretching, as illustrated in (B). The data show that HtpG suppresses entry into state X more strongly than the other states. Error bars represent the standard error calculated by assuming each population is independent.See also Figure S2.View Large Image Figure ViewerDownload Hi-res image Down" @default.
• W4310592040 created "2022-12-12" @default.
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• W4310592040 title "Direct observation of Hsp90-induced compaction in a protein chain" @default.
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