Insights into Small Heat Shock Protein and Substrate Structure during Chaperone Action Derived from Hydrogen/Deuterium Exchange and Mass Spectrometry

Abstract

Small heat shock proteins (sHSPs) and the related α-crystallins are ubiquitous chaperones linked to neurodegenerative diseases, myopathies, and cataract. To better define their mechanism of chaperone action, we used hydrogen/deuterium exchange and mass spectrometry (HXMS) to monitor conformational changes during complex formation between the structurally defined sHSPs, pea PsHsp18.1, and wheat TaHsp16.9, and the heat-denatured model substrates malate dehydrogenase (MDH) and firefly luciferase. Remarkably, we found that even when complexed with substrate, the highly dynamic local structure of the sHSPs, especially in the N-terminal arm (>70% exchange in 5 s), remains unchanged. These results, coupled with sHSP-substrate complex stability, indicate that sHSPs do not adopt new secondary structure when binding substrate and suggest sHSPs are tethered to substrate at multiple sites that are locally dynamic, a feature that likely facilitates recognition and refolding of sHSP-bound substrate by the Hsp70/DnaK chaperone system. Both substrates were found to be stabilized in a partially unfolded state that is observed only in the presence of sHSP. Furthermore, peptide-level HXMS showed MDH was substantially protected in two core regions (residues 95–156 and 228–252), which overlap with the MDH structure protected in the GroEL-bound MDH refolding intermediate. Significantly, despite differences in the size and structure of TaHsp16.9-MDH and PsHsp18.1-MDH complexes, peptide-level HXMS patterns for MDH in both complexes are virtually identical, indicating that stabilized MDH thermal unfolding intermediates are not determined by the identity of the sHSP. Small heat shock proteins (sHSPs) and the related α-crystallins are ubiquitous chaperones linked to neurodegenerative diseases, myopathies, and cataract. To better define their mechanism of chaperone action, we used hydrogen/deuterium exchange and mass spectrometry (HXMS) to monitor conformational changes during complex formation between the structurally defined sHSPs, pea PsHsp18.1, and wheat TaHsp16.9, and the heat-denatured model substrates malate dehydrogenase (MDH) and firefly luciferase. Remarkably, we found that even when complexed with substrate, the highly dynamic local structure of the sHSPs, especially in the N-terminal arm (>70% exchange in 5 s), remains unchanged. These results, coupled with sHSP-substrate complex stability, indicate that sHSPs do not adopt new secondary structure when binding substrate and suggest sHSPs are tethered to substrate at multiple sites that are locally dynamic, a feature that likely facilitates recognition and refolding of sHSP-bound substrate by the Hsp70/DnaK chaperone system. Both substrates were found to be stabilized in a partially unfolded state that is observed only in the presence of sHSP. Furthermore, peptide-level HXMS showed MDH was substantially protected in two core regions (residues 95–156 and 228–252), which overlap with the MDH structure protected in the GroEL-bound MDH refolding intermediate. Significantly, despite differences in the size and structure of TaHsp16.9-MDH and PsHsp18.1-MDH complexes, peptide-level HXMS patterns for MDH in both complexes are virtually identical, indicating that stabilized MDH thermal unfolding intermediates are not determined by the identity of the sHSP. The small heat shock proteins (sHSPs) 2The abbreviations used are: sHSPsmall heat shock proteinHXMShydrogen/deuterium exchange and mass spectrometryMDHmalate dehydrogenaseSECsize exclusion chromatographyPDBProtein Data BankLucluciferase. and related vertebrate α-crystallins are a ubiquitous class of molecular chaperones associated with diverse cellular activities (1Van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (389) Google Scholar, 2Haslbeck M. Franzmann T. Weinfurtner D. Buchner J. Nat. Struct. Mol. Biol. 2005; 12: 842-846Crossref PubMed Scopus (691) Google Scholar). In addition to attaining high levels of expression during high temperature stress, sHSPs are induced by other stresses (i.e. oxidative stress, heavy metals, ischemic injury) and are constitutive components of specific tissues in many different organisms. sHSPs have been found to modulate a wide range of biological processes, including cytoskeletal dynamics, cell differentiation, aging, and apoptosis (3Dierick I. Irobi J. De Jonghe P. Timmerman V. Ann. Med. (Basingstoke, UK). 2005; 37: 413-422Crossref PubMed Scopus (49) Google Scholar, 4Arrigo A.-P. Adv. Exp. Med. Biol. 2007; 594: 14-26Crossref PubMed Scopus (175) Google Scholar). Furthermore, expression and/or mutation of specific sHSPs is linked to neurodegenerative diseases, myopathies, and cataract (5Brady J.P. Garland D. Dublas-Tabor Y. Robison Jr., W.G. Groome A. Wawrousek E.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 884-889Crossref PubMed Scopus (304) Google Scholar, 6Sun Y. MacRae T.H. Cell Mol. Life Sci. 2005; 62: 2460-2476Crossref PubMed Scopus (414) Google Scholar, 7Bjoerkdahl C. Sjoegren M.J. Winblad B. Pei J.-J. J. Neurosci. Res. 2008; 86: 1342-1352Google Scholar), and sHSPs have been suggested to have therapeutic potential for amyotrophic lateral sclerosis (8Sharp P.S. Akbar M.T. Bouri S. Senda A. Joshi K. Chen H.-J. Latchman D.S. Wells D.J. de Belleroche J. Neurobiol. Dis. 2008; 30: 42-55Crossref PubMed Scopus (94) Google Scholar) and multiple sclerosis (9Ousman S.S. Tomooka B.H. van Noort J.M. Wawrousek E.F. O'Conner K. Hafler D.A. Sobel R.A. Robinson W.H. Steinman L. Nature. 2007; 448: 474-479Crossref PubMed Scopus (437) Google Scholar). The mechanism of sHSP chaperone action and interaction with substrates, therefore, has wide-ranging implications for understanding cellular stress and disease processes. small heat shock protein hydrogen/deuterium exchange and mass spectrometry malate dehydrogenase size exclusion chromatography Protein Data Bank luciferase. The sHSPs are defined by a signature α-crystallin domain of ∼100 amino acids, flanked by a short C-terminal extension and an N-terminal arm of variable length and divergent sequence (1Van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (389) Google Scholar). sHSP monomers range from ∼12 to 42 kDa, but in their native state, the majority of sHSPs form oligomers of 12 to >32 subunits. X-ray crystallographic structures of the 24-subunit Methanococcus jannaschii sHSP, MjHsp16.5 (10Kim K.K. Kim R. Kim S.-H. Nature. 1998; 394: 595-599Crossref PubMed Scopus (795) Google Scholar) and dodecameric Triticum aestivum (wheat) TaHsp16.9 (11Van Montfort R.L.M. Basha E. Friedrich K.L. Slingsby C. Vierling E. Nat. Struct. Biol. 2001; 8: 1025-1030Crossref PubMed Scopus (640) Google Scholar) reveal that the α-crystallin domain comprises a β-sandwich with topology identical to the Hsp90 co-chaperone p23, and participates in strand exchange to form a dimeric building block. A conserved IX(I/V) motif in the C-terminal extension makes essential oligomeric contacts by “patching” one edge of the α-crystallin β-sandwich. All of the N-terminal arms in the MjHsp16.5 structure were disordered (10Kim K.K. Kim R. Kim S.-H. Nature. 1998; 394: 595-599Crossref PubMed Scopus (795) Google Scholar), as were six in TaHsp16.9, but the remaining six had helical structure and were entwined to stabilize the oligomer (11Van Montfort R.L.M. Basha E. Friedrich K.L. Slingsby C. Vierling E. Nat. Struct. Biol. 2001; 8: 1025-1030Crossref PubMed Scopus (640) Google Scholar). Remarkably, although sHSPs are observed as stable oligomeric structures by many analytical techniques, kinetic studies reveal the oligomers undergo rapid subunit exchange (12Bova M.P. Ding L.-L. Horwitz J. Fung B.K.K. J. Biol. Chem. 1997; 272: 29511-29517Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 13Bova M.P. McHaourab H.S. Han Y. Fung B.K.K. J. Biol. Chem. 2000; 275: 1035-1042Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 14Sobott F. Benesch J.L.P. Vierling E. Robinson C.V. J. Biol. Chem. 2002; 277: 38921-38929Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), which is potentially critical to their function. The model for sHSP chaperone action has been developed from studies of diverse sHSPs and their interactions with multiple model substrates in vitro, and is supported by in vivo studies (1Van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (389) Google Scholar,2Haslbeck M. Franzmann T. Weinfurtner D. Buchner J. Nat. Struct. Mol. Biol. 2005; 12: 842-846Crossref PubMed Scopus (691) Google Scholar). sHSPs bind denaturing substrate proteins in an ATP-independent fashion and have a very high substrate capacity, binding up to an equal weight of some proteins. Proteins bound in the resulting high molecular weight sHSP-substrate complexes can be by the chaperone action of the Hsp70/DnaK by and in chaperones Vierling E. 2000; PubMed Scopus Google Scholar, A. C. Friedrich K.L. H.-J. Vierling E. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. S. Buchner J. P. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). with substrate is to binding sites the sHSP, which are by an of subunit by heat of the sHSP oligomer to a dimeric the chaperone mechanism of sHSPs a understanding of sHSPs and bind of the sHSP-substrate interaction is of the associated with protein by sHSP-substrate complexes (1Van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (389) Google Scholar, 2Haslbeck M. Franzmann T. Weinfurtner D. Buchner J. Nat. Struct. Mol. Biol. 2005; 12: 842-846Crossref PubMed Scopus (691) Google Scholar). Both the N-terminal arm and strand of the α-crystallin domain have been as sHSP substrate binding but overlap of binding sites with for sHSP E. Friedrich K.L. Vierling E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Basha E. Vierling E. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). is sHSP that formation of sHSP-substrate sHSP subunit exchange can in the presence of bound substrate (12Bova M.P. Ding L.-L. Horwitz J. Fung B.K.K. J. Biol. Chem. 1997; 272: 29511-29517Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, K.L. Vierling E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and the chaperone remains Vierling E. J. 1997; PubMed Scopus Google Scholar, M. S. T. M. Chen S. Buchner J. J. PubMed Scopus Google Scholar), although a that sites the sHSP N-terminal arm are protected in substrate complexes Res. 2007; PubMed Scopus Google Scholar). is of the The structure of substrates associated with sHSPs has been by and proteins that as the and but heat of and other proteins have been (1Van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (389) Google Scholar, R.A. C. D. C. J. Mol. Biol. 2002; PubMed Scopus Google Scholar, R.A. J. 2001; PubMed Scopus Google Scholar). that substrates bind when in an partially unfolded although both and unfolding intermediates have been as binding To sHSP-substrate we have hydrogen/deuterium exchange coupled with mass spectrometry to the conformational changes of sHSPs and model substrates in sHSP-substrate The to HXMS with complex in and with a to studies of not to other high techniques, and is used to and other complex structures J. M. M. E. Robinson C.V. Nature. 2005; PubMed Scopus Google Scholar, I. R. PubMed Scopus Google Scholar, L. J.M. V. Nat. Struct. Mol. Biol. 2007; PubMed Scopus Google Scholar). HXMS exchange of protein with in the The of exchange to and in Y. L. Proteins Struct. PubMed Scopus Google Scholar). of exchange for proteins in different can reveal differences in protein and by at the by differences can be to specific protein regions J. 2005; Scopus Google Scholar, C. B. M.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To in the of for studies we used TaHsp16.9 and related from PsHsp18.1, complexed with the chaperone substrates malate dehydrogenase (MDH) and firefly luciferase. sHSPs the structure of denaturing substrates and sHSPs bind substrate in a that to other malate dehydrogenase and firefly were from the and used Protein aestivum (wheat) TaHsp16.9 and were in and as Vierling E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Protein were determined the of for both proteins. The molecular of both proteins were by mass and substrate proteins were at and in the Protein were and for at The was a at a of The used in the was and for chromatography kDa, kDa, kDa, kDa, and of MDH and sHSPs by the by of sHSPs and MDH with a of of protein was and in to a of by addition of to the to The was to a of L. Mol. Cell 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), and as the at a of The protein was by a and for at a of in the were a coupled to a with a of for mass J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). were from the 12 a of at a of The and were in during the The was a mass in to The used in was Both mass and mass were used for The was used for exchange exchange were by the protein the and are in the All and were from the and were not for PubMed Scopus Google Scholar). an of of protein was of the exchange and by the with in to and in The of to the was by of protein with to a The of in were to for with protein exchange by a in were to a and All were of the were to a and to were at the is to that the of the was at is during the by as the In the temperature of the was at by of and the in an to exchange is that the of and temperature the exchange by of with that at and Y. L. Proteins Struct. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Y. J. 1997; PubMed Scopus Google Scholar). Furthermore, for during the can be a as by for were as for protein in the protein were to the protein with The was not used for the with the was by a and was used for protein spectrometry of were the with the of protein was with the The mass of was as the mass of the with the J. 1998; PubMed Scopus Google Scholar). To for the exchange of during the and the of only during the which the exchange an was A protein was by denaturing the sHSPs substrates in and for to mass The of was to Y. J. 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar), is the mass of the at a is the mass of the is the mass of the and is the of for All exchange have been with proteins from to and from to between sHSPs and of sHSP-substrate complexes, we complexes with MDH has been used as a model substrate for the chaperone K. T. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. K. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Mol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) and for sHSPs, including TaHsp16.9 and A. C. Friedrich K.L. H.-J. Vierling E. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. Friedrich K.L. Vierling E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). complexes were by TaHsp16.9 with MDH at an of at for a and These were of MDH from heat and a is TaHsp16.9 E. Friedrich K.L. Vierling E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of high molecular weight complexes MDH with sHSP at as observed by size exclusion chromatography at MDH was in the of sHSPs, the protein as by the in the MDH native In when the sHSPs are the majority of the sHSP to protein at the of the with a in the of TaHsp16.9 at to be a both sHSPs are differences in the sHSP-substrate complexes PsHsp18.1-MDH complexes to an size of kDa, TaHsp16.9-MDH complexes in the with E. Friedrich K.L. Vierling E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of TaHsp16.9 to MDH not complex size In virtually of the MDH and sHSP in the are the complex at the TaHsp16.9 at the of sHSP, with TaHsp16.9 with MDH is complexed with MDH and to during Both sHSP-substrate complexes are in size and/or the of the complex the of and the of the complexes suggest that TaHsp16.9 and have different of interaction with of sHSP in the monitor changes during sHSP-substrate complex we used the for as in protein was at for were to and to in The of both in the sHSP-substrate complexes can be by of their in The for and TaHsp16.9 in the presence of MDH are in In the native state, to both sHSPs of the the of at for both and the interactions between the sHSPs and MDH a by one to a for sHSP as and sHSP with but was not To was specific to complexes between sHSP and we complexes by with firefly for at 42 an sHSP to substrate of to from and the majority of both sHSP and are complexed TaHsp16.9 not form stable complexes with at temperature E. Friedrich K.L. Vierling E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), indicating interaction with substrate with PsHsp18.1, and was not used in we with was in in complex with at of that sHSP exchange is by interaction between sHSP and the of the the of complex formation with to the that were changes in of between and complexed sHSP, of mass changes at the was have at the for native TaHsp16.9 in the of substrate Friedrich K.L. Vierling E. PubMed Scopus Google Scholar). regions of the sHSP that are in oligomeric the N-terminal arm and C-terminal were the (>70% 5 s), the dynamic of the sHSP and the essential of the N-terminal for native TaHsp16.9 in with native and for both sHSPs with MDH by of of TaHsp16.9 and was for both proteins. The of native was to that observed for TaHsp16.9, with the rapid exchange in regions to form critical oligomeric of the for the complexes, even when a of 5 to complexes as observed by not not reveal differences with the native sHSPs was that sHSP in one mass we have been to mass the of of the interaction with substrate not formation of new stable structures the sHSP between sHSP and substrate, do substrate interactions to of the sHSP. of MDH in the heat and complex formation the structure of MDH as by we the in mass of MDH the of the in In to the sHSPs, MDH showed a of in both the PsHsp18.1-MDH and TaHsp16.9-MDH complexes In the native to the a MDH mass by during heat a new of MDH that an unfolded form of as a of of In the native form from the although a in the PsHsp18.1-MDH the MDH is the the of the from a of the of native to unfolded as the heat TaHsp16.9-MDH complexes, the native form was determined to be to the in MDH differences in MDH interaction with the two MDH was used to form complexes with TaHsp16.9, the of of the MDH native form was for both sHSPs the used for 5 s), a have of Y. L. Proteins Struct. PubMed Scopus Google Scholar). The that even the high mass of MDH only exchange the presence of some MDH secondary structure that the interaction of sHSP and MDH in the of sHSP and the MDH by HXMS showed that only the MDH native form was which with with the that the protein and of E. Friedrich K.L. Vierling E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the MDH form observed by HXMS in complexes is stable only in the presence of the sHSP. were to of an unfolded form of in complex with In the of sHSP at temperature of in complex with of for unfolded form is only stable in the presence of the sHSP. of in MDH with of MDH are to in the sHSP-substrate complexes, was of to was to of a specific of MDH and were differences between and To complexes were to peptide-level of MDH with was and were used in the of the sequence from to amino acids, with an length of 12 were to the with of the of sHSP to for the to in the of sHSP the is that of MDH complexed with sHSPs is different that observed for native and that is the protein are two core regions of 95–156 and which are protected exchange heat in the presence of sHSP. mass of and the regions are in in the of regions from exchange is MDH is in the presence of sHSP. the are of regions remains even MDH was in the presence of sHSP. the exchange at for 5 s), the of exchange for core regions is substantially the exchange for structure were Y. L. Proteins Struct. PubMed Scopus Google Scholar). To in a was in which MDH in the presence of was to for from 5 to A of exchange for the in the two protected regions is in be that of the partially protected even is for and both of which A is that the of the partially unfolded MDH not with that of the native protein and The and of MDH a of exchange in the native In the sHSP-bound state, both regions their exchange for a at which only A from and with the exchange is that the MDH patterns were was complexed with TaHsp16.9, despite the the complexes in the not only the mass but the of the mass for to be sHSPs of HXMS to peptide-level conformational of both sHSP and substrate in sHSP-substrate complexes new the mechanism of sHSP chaperone A was to peptide-level changes in in TaHsp16.9 with denaturing have for TaHsp16.9, and for PsHsp18.1, that of the N-terminal arm and C-terminal extension are highly (>70% in 5 s), despite the of both of regions in oligomer (1Van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (389) Google Scholar, Montfort R.L.M. Basha E. Friedrich K.L. Slingsby C. Vierling E. Nat. Struct. Biol. 2001; 8: 1025-1030Crossref PubMed Scopus (640) Google Scholar). the N-terminal arm as for substrate although substrate interaction with multiple sites the sHSP is likely E. Friedrich K.L. Vierling E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Basha E. Vierling E. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, M. A. S. E. T. Buchner J. J. Mol. Biol. PubMed Scopus Google Scholar). that with the substrate at a the dynamic of the N-terminal which substrate recognition amino and/or The of in the peptide-level in both the highly regions of the sHSP, as as the α-crystallin is with the that interactions are highly dynamic, with an the by protein which from formation of stable structures Y. L. Proteins Struct. PubMed Scopus Google Scholar), sHSP-substrate formation not of stable secondary although interactions with substrate to be dynamic, to be a local as complexes are stable and MDH be to sHSP to A. C. Friedrich K.L. H.-J. Vierling E. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K.L. Vierling E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), indicating that substrates are tethered at multiple sites which of substrate to sHSPs can substrates, but at the can substrates to be to the Hsp70/DnaK to the sHSPs, both MDH and showed a in during and with the sHSP. Both substrates highly protected of the remaining of sHSP-bound substrate from of a binding between sHSP and The that was not observed the sHSP that by a binding is not a to the for In has been to the of MDH when bound to PubMed Scopus Google Scholar, J. S. Nat. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar). In MDH was to the structure of MDH in the bound In Chen J. S. Nat. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar) HXMS to monitor the of MDH refolding by In that were protected from exchange when MDH was bound to addition of MDH was from the of and a of was the was to the structure of MDH in the bound HXMS studies for as as the of sHSP binding to MDH observed in that at the of the observed in substrate and of that the sHSP-bound MDH and are partially protected exchange is in with studies that have sHSPs bind substrate in some form of state (1Van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (389) Google Scholar, R.A. J. 2001; PubMed Scopus Google Scholar, R.A. M. Protein Struct. Mol. 2000; PubMed Scopus Google Scholar). we were to peptide-level changes in a substrate HXMS that the bound MDH structure is not the as in the native The highly stable and are only the core in the of sHSP, stable heat-denatured can be by size exclusion chromatography were to stable by during heat of MDH MDH unfolding is and is short to be by the the sHSP a the heat is that the regions of sHSP-bound MDH the exchange overlap with found in GroEL-bound MDH J. S. Nat. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar). a of exchange between native MDH and that protected 95–156 and which secondary structure and to the MDH were the protected exchange when MDH was bound to J. S. Nat. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar). In studies with the substrate protein is and a of to binding and in MDH was in the presence of sHSP. in the bound state, the substrate structure be the mechanism of is different from that of In the only one of substrate at a and substrate is the PubMed Scopus Google Scholar). has two GroEL-bound substrate not have to with other substrate is in to the which likely of multiple of both sHSP and the the the conformational of the substrate In the of remains to be determined MDH is to the as with MDH in of the from and the sHSP to MDH to substrate TaHsp16.9-MDH and PsHsp18.1-MDH complexes are a different of sHSP and peptide-level HXMS of MDH in two complexes was which of MDH secondary and that the identity of the sHSP not the substrate unfolding The in complex size with and TaHsp16.9 be by the that thermal unfolding is a kinetic very in and TaHsp16.9 have different for different substrates Vierling E. J. 1997; PubMed Scopus Google Scholar, E. B. Vierling E. J. PubMed Scopus (94) Google Scholar). MDH a partially unfolded state, sHSP as as other of partially unfolded MDH for with is in of MDH is likely to be resulting in a complex size with complexes with The that the unfolding of MDH in the presence of sHSP is not determined by the identity of sHSP is in with a of the interaction of α-crystallin with by R.A. C. D. C. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). observed refolding of from by and the presence of α-crystallin not the refolding sHSPs are to be chaperones that do not the of protein critical sHSP-substrate HXMS can reveal conformational of the sHSP-substrate binding To we are sHSP-substrate contacts is to define the of sHSP-substrate interactions that to the in of substrate for proteins as and substrate binding is to sHSPs can diverse in stress, and with