SUBSTRATE BINDING SITE FLEXIBILITY OF SMALL HEAT SHOCK PROTEINS AND FACTORS CONTRIBUTING TO EFFICIENT CHAPERONE ACTIVITY

Persistent Link:
http://hdl.handle.net/10150/193550
Title:
SUBSTRATE BINDING SITE FLEXIBILITY OF SMALL HEAT SHOCK PROTEINS AND FACTORS CONTRIBUTING TO EFFICIENT CHAPERONE ACTIVITY
Author:
Jaya, Nomalie Naomi
Issue Date:
2009
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
sHSPs maintain partially denaturing substrates in a soluble sHSP-substrate complex. The heterogeneous interaction between sHSPs and substrate within the complex has prevented a detailed study of the mechanism of sHSP substrate protection. Here, purified sHSPs and heat sensitive substrates were used to investigate the mechanism of sHSP chaperone action. Results presented provide new insights into how sHSPs recognize substrates, the architecture of the sHSP-substrate complex and factors contributing to chaperone efficiency.Direct evidence defining the role of the sHSP N-terminal arm and alpha crystallin domain in sHSP-substrate interactions is limited. A photoactivatable probe was site- specifically incorporated into PsHsp18.1, and cross-linking to substrate in sHSP-substrate complexes was quantified. The structurally flexible N-terminal arm of PsHsp18.1 makes strong contacts with both substrates tested, however differences in interaction were seen in the conserved alpha crystallin domain. Regions on the sHSP showing the strongest cross-links to substrates are buried within the dodecamer, supporting the model that the sHSP oligomer undergoes rearrangement or dissociation prior to substrate interactions.The arrangement of sHSPs and substrates whithin the complex is poorly defined. Limited proteolysis and chemical modification was combined with mass spectrometry to probe the sHSP-substrate complex using multiple sHSPs and substrates. This analysis reveals that a similar partially-denatured form of substrate is protected in the complex irrespective of sHSP identity. Further, sHSP in the complex is protected from proteolysis for a longer time compared to free sHSP. These data suggest that sHSPs and substrate are distributed both internally and on the periphery of the sHSP-substrate complex.Exact properties of the sHSP N-terminal arm contributing to protection are poorly defined. A molecular dynamics (MD) study was designed to test the hypothesis that the N-terminal arm could assume multiple conformations that can readily interact with denaturing substrates. Preliminary data suggest that at increased temperatures amino acids in the N-terminal arm form specific clusters which could act as substrate interaction sites. MD simulations, mutagenesis and altering the kinetics of substrate aggregation suggest that the conformational space occupied by the N-terminal arm at increased temperatures, along with flexibility and rate of substrate aggregation contribute to differences in chaperone efficiency.
Type:
text; Electronic Dissertation
Keywords:
Alpha-crystallin; Chaperones; Cross-linking; Intrinsic disorder; Mass spectrometry; Protein-protein interactions
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Biochemistry & Molecular Biophysics; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Vierling, Elizabeth
Committee Chair:
Vierling, Elizabeth

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleSUBSTRATE BINDING SITE FLEXIBILITY OF SMALL HEAT SHOCK PROTEINS AND FACTORS CONTRIBUTING TO EFFICIENT CHAPERONE ACTIVITYen_US
dc.creatorJaya, Nomalie Naomien_US
dc.contributor.authorJaya, Nomalie Naomien_US
dc.date.issued2009en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractsHSPs maintain partially denaturing substrates in a soluble sHSP-substrate complex. The heterogeneous interaction between sHSPs and substrate within the complex has prevented a detailed study of the mechanism of sHSP substrate protection. Here, purified sHSPs and heat sensitive substrates were used to investigate the mechanism of sHSP chaperone action. Results presented provide new insights into how sHSPs recognize substrates, the architecture of the sHSP-substrate complex and factors contributing to chaperone efficiency.Direct evidence defining the role of the sHSP N-terminal arm and alpha crystallin domain in sHSP-substrate interactions is limited. A photoactivatable probe was site- specifically incorporated into PsHsp18.1, and cross-linking to substrate in sHSP-substrate complexes was quantified. The structurally flexible N-terminal arm of PsHsp18.1 makes strong contacts with both substrates tested, however differences in interaction were seen in the conserved alpha crystallin domain. Regions on the sHSP showing the strongest cross-links to substrates are buried within the dodecamer, supporting the model that the sHSP oligomer undergoes rearrangement or dissociation prior to substrate interactions.The arrangement of sHSPs and substrates whithin the complex is poorly defined. Limited proteolysis and chemical modification was combined with mass spectrometry to probe the sHSP-substrate complex using multiple sHSPs and substrates. This analysis reveals that a similar partially-denatured form of substrate is protected in the complex irrespective of sHSP identity. Further, sHSP in the complex is protected from proteolysis for a longer time compared to free sHSP. These data suggest that sHSPs and substrate are distributed both internally and on the periphery of the sHSP-substrate complex.Exact properties of the sHSP N-terminal arm contributing to protection are poorly defined. A molecular dynamics (MD) study was designed to test the hypothesis that the N-terminal arm could assume multiple conformations that can readily interact with denaturing substrates. Preliminary data suggest that at increased temperatures amino acids in the N-terminal arm form specific clusters which could act as substrate interaction sites. MD simulations, mutagenesis and altering the kinetics of substrate aggregation suggest that the conformational space occupied by the N-terminal arm at increased temperatures, along with flexibility and rate of substrate aggregation contribute to differences in chaperone efficiency.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectAlpha-crystallinen_US
dc.subjectChaperonesen_US
dc.subjectCross-linkingen_US
dc.subjectIntrinsic disorderen_US
dc.subjectMass spectrometryen_US
dc.subjectProtein-protein interactionsen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineBiochemistry & Molecular Biophysicsen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorVierling, Elizabethen_US
dc.contributor.chairVierling, Elizabethen_US
dc.contributor.committeememberVierling, Elizabethen_US
dc.contributor.committeememberBandarian, Vaheen_US
dc.contributor.committeememberLittle, Johnen_US
dc.contributor.committeememberMontfort, Williamen_US
dc.contributor.committeememberWysocki, Vickien_US
dc.identifier.proquest10615en_US
dc.identifier.oclc659752384en_US
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