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• W1996444327 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. 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 (376) Google Scholar, 2Haslbeck M. Franzmann T. Weinfurtner D. Buchner J. Nat. Struct. Mol. Biol. 2005; 12: 842-846Crossref PubMed Scopus (663) 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 (173) 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 (297) Google Scholar, 6Sun Y. MacRae T.H. Cell Mol. Life Sci. 2005; 62: 2460-2476Crossref PubMed Scopus (396) 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 (90) 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 (419) Google Scholar). The mechanism of sHSP chaperone action and interaction with substrates, therefore, has wide-ranging implications for understanding cellular stress and disease processes.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 (376) 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 (783) 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 (624) 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 (783) 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 (624) 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 (267) 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 (215) 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 (167) 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 (376) Google Scholar,2Haslbeck M. Franzmann T. Weinfurtner D. Buchner J. Nat. Struct. Mol. Biol. 2005; 12: 842-846Crossref PubMed Scopus (663) 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 refolded by the ATP-dependent chaperone action of the Hsp70/DnaK system, assisted by Hsp100/ClpB and GroEL in cells/compartments where these latter chaperones co-occur (15Lee G.J. Vierling E. Plant Physiol. 2000; 122: 189-197Crossref PubMed Scopus (365) Google Scholar, 16Mogk A. Schlieker C. Friedrich K.L. Schoenfeld H.-J. Vierling E. Bukau B. J. Biol. Chem. 2003; 278: 31033-31042Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 17Veinger L. Diamant S. Buchner J. Goloubinoff P. J. Biol. Chem. 1998; 273: 11032-11037Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Interaction with substrate is believed to involve hydrophobic binding sites on the sHSP, which are exposed by an increased rate of subunit exchange, by heat or phosphorylation-induced shift of the sHSP oligomer equilibrium to a dimeric form, or through more subtle structural rearrangements.Defining the chaperone mechanism of sHSPs requires a more complete understanding of how sHSPs recognize and bind substrates. Detailed study, however, of the sHSP-substrate interaction is challenging, because of the difficulties associated with investigating heterogeneous protein mixtures exemplified by sHSP-substrate complexes (1Van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (376) Google Scholar, 2Haslbeck M. Franzmann T. Weinfurtner D. Buchner J. Nat. Struct. Mol. Biol. 2005; 12: 842-846Crossref PubMed Scopus (663) Google Scholar). Both the N-terminal arm and β-4 strand of the α-crystallin domain have been implicated as sHSP substrate binding sites, but overlap of proposed binding sites with structural elements required for sHSP oligomerization (and therefore structural integrity) complicates data interpretation (18Basha E. Friedrich K.L. Vierling E. J. Biol. Chem. 2006; 281: 39943-39952Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 19Giese K.C. Basha E. Catague B.Y. Vierling E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18896-18901Crossref PubMed Scopus (63) Google Scholar). Virtually nothing is known about sHSP structural rearrangements that must accompany formation of sHSP-substrate complexes. sHSP subunit exchange can continue 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 (267) Google Scholar, 20Friedrich K.L. Giese K.C. Buan N.R. Vierling E. J. Biol. Chem. 2004; 279: 1080-1089Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and the chaperone remains protease-accessible (21Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (653) Google Scholar, 22Haslbeck M. Walke S. Stromer T. Ehrnsperger M. White H.E. Chen S. Saibil H.R. Buchner J. EMBO J. 1999; 18: 6744-6751Crossref PubMed Scopus (380) Google Scholar), although a recent study indicates that protease sites on the sHSP N-terminal arm are protected in substrate complexes (23Aquilina J.A. Watt S.J. Biochem. Biophys. Res. Commun. 2007; 353: 1115-1120Crossref PubMed Scopus (33) Google Scholar). There is more, though still limited, information about organization of the denatured substrate. The structure of substrates associated with sHSPs has been monitored by fluorescence dye binding, intrinsic fluorescence, CD, 1H NMR, and spin labeling, primarily using proteins that aggregate upon reduction, such as the insulin β-chain and α-lactalbumin, but heat aggregation of rhodanese, carbonic anhydrase, and other proteins have also been investigated (1Van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (376) Google Scholar, 24Carver J.A. Lindner R.A. Lyon C. Canet D. Hernandez H. Dobson C.M. Redfield C. J. Mol. Biol. 2002; 318: 815-827Crossref PubMed Scopus (100) Google Scholar, 25Lindner R.A. Treweek T.M. Carver J.A. Biochem. J. 2001; 354: 79-87Crossref PubMed Scopus (80) Google Scholar). Results generally agree that substrates bind when in an aggregation-prone, partially unfolded molten globule form, although both early and late unfolding intermediates have been identified as binding structures.To gain more detailed insight into sHSP-substrate interactions, we have employed solution phase hydrogen/deuterium exchange (HX) coupled with mass spectrometry (MS) to investigate the conformational changes of sHSPs and model substrates in sHSP-substrate complexes. The ability to perform HXMS with complex mixtures in physiological buffers and with minimal material make this a valuable approach to structural studies of systems not amenable to other high resolution techniques, and it is now being used to probe amyloid and other complex structures (26Carulla N. Caddy G.L. Hall D.R. Zurdo J. Gairi M. Feliz M. Giralt E. Robinson C.V. Dobson C.M. Nature. 2005; 436: 554-558Crossref PubMed Scopus (299) Google Scholar, 27Kheterpal I. Cook K.D. Wetzel R. Methods Enzymol. 2006; 413: 140-166Crossref PubMed Scopus (29) Google Scholar, 28Wu Z. Wagner M.A. Zheng L. Parks J.S. Shy III, J.M. Smith J.D. Gogonea V. Hazen S.L. Nat. Struct. Mol. Biol. 2007; 14: 861-868Crossref PubMed Scopus (181) Google Scholar). HXMS monitors exchange of protein backbone amide hydrogens with deuterium in the D2O solvent. The rate of exchange depends on amide hydrogen access to D2O and involvement in internal hydrogen bonds (29Bai Y. Milne J.S. Mayne L. Englander S.W. Proteins Struct. Funct. Genet. 1993; 17: 75-86Crossref PubMed Scopus (1743) Google Scholar). Comparison of exchange rates for proteins in different states can reveal differences in protein conformation, and importantly, by measuring HX at the peptide level by MS, structural differences can be located to specific protein regions (30Komives E.A. Int. J. Mass Spectrom. 2005; 240: 285-290Crossref Scopus (24) Google Scholar, 31Rist W. Graf C. Bukau B. Mayer M.P. J. Biol. Chem. 2006; 281: 16493-16501Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 32Shi J. Koeppe J.R. Komives E.A. Taylor P. J. Biol. Chem. 2006; 281: 12170-12177Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar).To enable data interpretation in the context of structure, for our studies we used TaHsp16.9 and its closely related homolog from Pisum sativum (pea), PsHsp18.1, complexed with the well-characterized chaperone substrates malate dehydrogenase (MDH) and firefly luciferase. Results here provide novel insights into how sHSPs affect the structure of denaturing substrates and how sHSPs bind substrate in a manner that may facilitate release to other chaperones.MATERIALS AND METHODSPig heart mitochondrial malate dehydrogenase (Roche Applied Science, PDB 1MLD) and firefly luciferase (Promega, PDB 1LCI) were purchased from the manufacturer and used without further purification.Protein Purification—Triticum aestivum (wheat) TaHsp16.9 (PDB 1GME) and Pisum sativum (pea) PsHsp18.1 (GenBank™ accession no. P19243) were expressed in Escherichia coli and purified as described previously (33Lee G.J. Pokala N. Vierling E. J. Biol. Chem. 1995; 270: 10432-10438Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Protein concentrations were determined using the calculated extinction coefficient of E280 = 16500 m-1 cm-1 for both proteins. The expected molecular masses of both proteins were confirmed by mass spectrometry.Size Exclusion Chromatography (SEC)—sHSPs and substrate proteins were incubated at concentrations, temperatures, and times specified in the figure legends or text. Protein mixtures were cooled on ice after heating and centrifuged for 15 min at 13,000 rpm. The supernatant (100 μl) was loaded onto a Bio-Sil SEC 400-5 column (Bio-Rad) at a flow rate of 1 ml/min. The mobile phase used in all the experiments was 25 mm sodium phosphate and 150 mm KCl, pH 7.5. Standards for chromatography were: thyroglobulin 670 kDa, γ-globulin 158 kDa, ovalumin 44 kDa, carbonic anhydrase 29 kDa, and myoglobin 17 kDa (Bio-Rad).Peptide Mapping of MDH and sHSPs by HPLC-Tandem Mass Spectrometry—To map the peptides produced by digestion of sHSPs and MDH with pepsin, a total of 100 pmol of protein stock was diluted into 25 mm sodium phosphate and 150 mm KCl in H2O (pH 7.5) to a final concentration of 20 μm, followed by addition of 25% formic acid to adjust the pH to pH 2.5 (by pH paper). The sample was applied to a column of immobilized pepsin (2 mm × 50 mm, packed in-house (34Wang L. Pan H. Smith D.L. Mol. Cell Proteom. 2002; 1: 132-138Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), using water and 0.05% trifluoroacetic acid as the mobile phase at a flow rate of 50 μl/min. The protein digest was collected by a micropeptide trap (Michrome BioResources, Auburn, CA) and washed for 2 min at a flow rate of 300 μl/min. Peptides in the trap were then eluted onto a microbore C-18 HPLC column (1 mm × 50 mm, Micro-Tech Scientific, Vista, CA) coupled to a Waters micro Q-TOF (Milford, MA) with a typical ESI voltage of 3 kV for accurate parent mass measurements (35Cheng G. Wysocki V.H. Cusanovich M.A. J. Am. Soc. Mass Spectrom. 2006; 17: 1518-1525Crossref PubMed Scopus (7) Google Scholar, 36Cheng G. Cusanovich M.A. Wysocki V.H. Biochemistry. 2006; 45: 11744-11751Crossref PubMed Scopus (8) Google Scholar). Peptides were eluted from the column over 12 min. using a gradient of 15–45% acetonitrile at a flow rate of 50 μl/min. The micropeptide trap and HPLC column were immersed in ice water during the entire process. The same experiment was repeated using a Finnigan LCQ Classic quadrupole ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) in data-dependent mode to acquire product (tandem) MS spectra. The typical ESI voltage used in LCQ was 4.5 kV. Both parent mass and tandem mass spectra were used for peptide identification. The Waters micro Q-TOF was subsequently used for all H/D exchange measurements.Hydrogen/Deuterium Exchange (HX)—HX exchange experiments were initiated by diluting the protein sample (∼20-fold) into the labeling solution (D2O, 25 mm sodium phosphate and 150 mm KCl, pD 7.5). Incubation times are specified in the text. All pH and pD values reported were taken directly from the pH meter and were not corrected for isotope effects (37Englander J.J. Rogero J.R. Englander S.W. Anal. Biochem. 1985; 147: 234-244Crossref PubMed Scopus (144) Google Scholar). At each time point, an aliquot of 200 μl of protein was taken out of the exchange tube and quenched by mixing the solution with 25% D2 formic acid in D2O to pD 2.5 and frozen in liquid nitrogen. The amount of formic acid required to achieve the desired pD was first estimated by titrating 20 ml of protein buffer with 25% formic acid to pH 2.5 using a pH meter. The required aliquots of 25% D2 formic acid in D2O were then added to individual quenching tubes for direct mixing with protein exchange samples, followed by a pH reading using pH paper. After freezing in liquid N2, quenched samples were transferred to a -80 °C freezer and stored until analysis. All samples were analyzed within 24 h of the HX experiments. Frozen samples were transferred to a dry ice container and removed directly prior to LC-MS analysis. Individual 200-μl samples were defrosted within <1 min at 37 °C before loading directly onto the pepsin column. It is important to note that the pH of the quenched samples was kept at ∼pH 2.5. This pH is maintained during the HPLC step by using 0.05% trifluoroacetic acid as the modifier. In addition, the temperature of the whole digestion/separation setup was maintained at ∼4 °C, by immersion of peptide/protein trap, LC column, and the connecting tubings in an ice bath to further decrease exchange rate. It is well documented that the combination of low pH and low temperature decreases the exchange reaction rate by five orders of magnitude compared with that at neutral pH and 25 °C (29Bai Y. Milne J.S. Mayne L. Englander S.W. Proteins Struct. Funct. Genet. 1993; 17: 75-86Crossref PubMed Scopus (1743) Google Scholar, 37Englander J.J. Rogero J.R. Englander S.W. Anal. Biochem. 1985; 147: 234-244Crossref PubMed Scopus (144) Google Scholar, 38Smith D.L. Deng Y. Zhang Z. J. Mass Spectrom. 1997; 32: 135-146Crossref PubMed Scopus (383) Google Scholar). Furthermore, correction for back-exchange during the LC-MS step can be done using a fully deuterated controlled sample, as described below.HX Analysis by HPLC-ESI—Samples for peptide analysis were treated as described for unlabeled protein in the peptide mapping section. Intact protein samples were analyzed similarly to the protein digest, with three exceptions. The pepsin column was not used for the MS experiment with intact protein, the micropeptide trap was replaced by a microprotein trap (Michrom BioResources), and 60% acetonitrile was used for protein elution. Mass spectrometry analyses of all samples within each comparison set were done on the same day with the same instrumental conditions. Deconvolution of intact protein spectra was performed with the program MaxEnt1 (Waters, MA). The mass of each peptide was taken as the centroid mass of the isotopic envelope with the program MagTran (39Zhang Z. Marshall A.G. J. Am. Soc. Mass Spectrom. 1998; 9: 225-233Crossref PubMed Scopus (444) Google Scholar).To account for the exchange of deuterium during the HPLC step (back exchange), and the use of only 20-fold excess D2O during the labeling step, which limits the forward exchange reaction, an experimental correction was applied. A 100% deuterated protein control (100D reference) was prepared by fully denaturing the sHSPs or substrates in 6 m GuDCl and diluting 20-fold into D2O for >24 h prior to mass determination. The corrected extent of deuterium incorporation was then calculated according to Equation 1 (38Smith D.L. Deng Y. Zhang Z. J. Mass Spectrom. 1997; 32: 135-146Crossref PubMed Scopus (383) Google Scholar, 40Wales T.E. Engen J.R. Mass Spectrom. Rev. 2006; 25: 158-170Crossref PubMed Scopus (669) Google Scholar), m=mexp-m0&x0025;m100&x0025;-m0&x0025;×N(Eq. 1) where mexp is the experimental centroid mass of the peptide at a certain time point, m0% is the centroid mass of the undeuterated control, m100% is the centroid mass of the 100% deuterated control, and N is the number of amide hydrogens for each protein/peptide characterized. All exchange results presented here have been corrected, with intact proteins correction factors ranging from 85 to 90%, and peptide correction factors ranging from 75 to 90%.RESULTSComplex Formation between sHSPs and Substrate—To investigate structural properties of sHSP-substrate complexes, we prepared complexes with thermally denatured MDH. MDH has been used extensively as a model substrate for the chaperone GroEL (41Farr G.W. Furtak K. Rowland M.B. Ranson N.A. Saibil H.R. Kirchhausen T. Horwich A.L. Cell. 2000; 100: 561-573Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 42Horst R. Bertelsen E.B. Fiaux J. Wider G. Horwich A.L. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12748-12753Crossref PubMed Scopus (100) Google Scholar, 43Fenton W.A. Horwich A.L. Quart. Rev. Biophys. 2003; 36: 229-256Crossref PubMed Scopus (127) Google Scholar, 44Elad N. Farr G.W. Clare D.K. Orlova E.V. Horwich A.L. Saibil H.R. Mol. Cell. 2007; 26: 415-426Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and for sHSPs, including TaHsp16.9 and PsHsp18.1 (16Mogk A. Schlieker C. Friedrich K.L. Schoenfeld H.-J. Vierling E. Bukau B. J. Biol. Chem. 2003; 278: 31033-31042Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 18Basha E. Friedrich K.L. Vierling E. J. Biol. Chem. 2006; 281: 39943-39952Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). sHSP-MDH complexes were formed by incubating either 24 μm PsHsp18.1 or TaHsp16.9 with MDH at an sHSP/MDH molar ratio of 2.4:1 or 3:1, respectively, at 45 °C for 0–120 min (Fig. 1, a and b). These sHSP/MDH ratios were chosen because they afford complete protection of MDH from heat insolubilization, and on a molar basis PsHSP18.1 is more effective than TaHsp16.9 (18Basha E. Friedrich K.L. Vierling E. J. Biol. Chem. 2006; 281: 39943-39952Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Accumulation of high molecular weight complexes comprising MDH with either sHSP increased over time at 45 °C as observed by size exclusion chromatography performed at room temperature. When MDH was heated in the absence of sHSPs, the protein aggregated, as shown by the gradual decrease in the MDH native peak (Fig. 1c). In contrast, when the sHSPs are heated alone, the majority of the sHSP elutes identically to unheated protein at the position of the dodecamer, with a minor fraction in the case of TaHsp16.9 eluting at position predicting to be a dimer (supplemental Fig. S1).Although both sHSPs fully protect MDH, there are significant differences in the sHSP-substrate complexes formed. PsHsp18.1-MDH complexes eluted later, corresponding to an apparent size of 600–700 kDa, while TaHsp16.9-MDH complexes eluted in the void volume (>1000 kDa), consistent with previous observations (18Basha E. Friedrich K.L. Vierling E. J. Biol. Chem. 2006; 281: 39943-39952Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Increasing the ratio of TaHsp16.9 to MDH did not reduce apparent complex size (not shown). In addition, while virtually all of the MDH and sHSP in the PsHsp18.1 samples are incorporated into the complex peak after 2 h at 45°C, after the same treatment, significant TaHsp16.9 elutes at the position of free sHSP, with no change on further heating (not shown). Thus, TaHsp16.9 either initially associates less effectively with MDH or is less stably complexed with MDH and dissociates prior to or during chromatography. Both sHSP-substrate complexes are heterogeneous in size and/or shape, based on the broadness of the complex peak. Altogether the stoichiometry of protection and the nature of the complexes formed suggest that TaHsp16.9 and PsHsp18.1 have somewhat different modes of interaction with MDH.HX of sHSP in the sHSP-Substrate Complex—To monitor structural changes during heat-induced sHSP-substrate complex formation, we used the protocol for HX as diagramed in Fig. 2a. Each protein mixture was heated at 45 °C for 0–120 min. Samples were removed every 30 min, cooled to 25 °C, and then subjected to 5-s pulse labeling in D2O. The HX information of both partners in the sHSP-substrate complexes can be monitored simultaneously by MS, because of their significant difference in monomeric mass. The HX data for PsHsp18.1 and TaHsp16.9 in the presence of MDH are shown in Fig. 2b. In the native state, prior to heating, both sHSPs exchanged 58% of backbone hydrogens. Surprisingly, throughout the heating time course, the extent of HX remained at 58% for both PsHsp18.1 and TaHsp16.9. If the interactions between the sHSPs and MDH leave a “fingerprint” by HX, one would expect to observe a two-population pattern for sHSP as more and more sHSP interacts with heat-denaturing MDH, but this was not observed.FIGURE 2Global HXMS of sHSP and substrate. a, experimental scheme for examining HX during sHSP-MDH complex formation. Concentrations of sHSP and MDH are as in Fig. 1. b, mass spectra of sHSP global exchange pattern as a function of time monitored by HXMS. Left, PsHsp18.1 in the presence of MDH. Three populations of PsHsp18.1 were present in the sample used in the experiment: unmodified protein, protein with N-terminal methylation and a very minor fraction of N-terminally acetylated protein. All forms bound substrate equally well (not shown). Right, TaHsp16.9 in the presence of MDH. Two populations of TaHsp16.9 were present in the protein used in the experiment: unmodified protein and a very minor fraction of N-terminally acetylated protein. c, mass spectra of MDH global HX pattern as a function of time. Left, MDH thermal unfolding in the presence of PsHsp18.1. Right, MDH thermal unfolding in the presence of TaHsp16.9.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To test if this result was specific to complexes between sHSP and MDH, we preformed HX on complexes formed by heating PsHsp18.1 together with firefly luciferase (Luc) for 7 min at 42 °C. At an sHSP to substrate ratio of 4 to 1, PsHsp18.1 fully protects Luc from insolubilization, and the vast majority of both sHSP and Luc are complexed (supplemental Fig. S2a). TaHsp16.9 does not protect or form stable complexes with Luc at this temperature (18Basha E. Friedrich K.L. Vierling E. J. Biol. Chem. 2006; 281: 39943-39952Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), again indicating differential interaction with substrate compared with PsHsp18.1, and was therefore not used in this experiment. As we saw with MDH, there was no change in total" @default.
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• W1996444327 date "2008-09-01" @default.
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• W1996444327 title "Insights into Small Heat Shock Protein and Substrate Structure during Chaperone Action Derived from Hydrogen/Deuterium Exchange and Mass Spectrometry" @default.
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• W1996444327 cites W1790977108 @default.
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• W1996444327 cites W1876533241 @default.
• W1996444327 cites W1966572509 @default.
• W1996444327 cites W1969249762 @default.
• W1996444327 cites W1969650644 @default.
• W1996444327 cites W1971997528 @default.
• W1996444327 cites W1975315404 @default.
• W1996444327 cites W1976602670 @default.
• W1996444327 cites W1981542877 @default.
• W1996444327 cites W1983502971 @default.
• W1996444327 cites W1998601781 @default.
• W1996444327 cites W2007363410 @default.
• W1996444327 cites W2007780468 @default.
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• W1996444327 cites W2022038675 @default.
• W1996444327 cites W2033104365 @default.
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• W1996444327 doi "https://doi.org/10.1074/jbc.m802946200" @default.
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