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Title page for ETD etd-01162008-104354

Type of Document Dissertation
Author Shi, Jian
URN etd-01162008-104354
Title A study of small heat shock proteins structure and function by cryo-electron microscopy.
Degree PhD
Department Molecular Physiology and Biophysics
Advisory Committee
Advisor Name Title
Albert H. Beth Committee Chair
Anne Kenworthy Committee Member
Charles R. Sanders, II Committee Member
Jens Meiler Committee Member
Roger J. Colbran Committee Member
  • small heat shock protein
  • Hsp16.5
  • cataract
  • molecular chaperone
  • single particle reconstruction
  • cryo electron microscopy
Date of Defense 2007-12-10
Availability unrestricted
Small heat shock proteins (sHSPs) are a ubiquitous family of chaperones that protect unfolded proteins from irreversible aggregation in the cell. Human sHSPs are associated with the pathology of a variety of diseases. The high resolution structures of mammalian members of the sHSP family have not yet been achieved presumably because they form polydisperse oligomers. The engineered variants of monodisperse Hsp16.5 adapt to diverse quaternary structures, therefore serve as a model system for mammalian sHsps. In this work, we have combined biophysical approaches, cryoEM single particle reconstruction and spin labeling EPR spectroscopy to study various forms of engineered Hsp16.5 and their complexes with substrates.

Our studies show that sequence variation in the N-terminal region of Hsp16.5 can dramatically influence its oligomeric structure. The oligomeric plasticity may result from the flexible linker region in the C-terminus, which allows two dimers to interact in a continuum of angles. A hypothetical model proposed for a polydisperse Hsp16.5 variant provides a feasible explanation for the polydispersity of human sHSPs. Our results suggest substrates are protected inside the sHSP oligomer through interactions with the N-termini and α-crystallin domain shell. For polydisperse and expanded oligomers, we hypothesize that increased volume and greater access to the substrate binding sites contribute to enhanced binding ability. Further, our knowledge from this study sheds light on the roles of sHSPs in human disease. Early onset cataractogenesis, related to an Arg mutation in human α-crystallin, may result from hyperactivity of the mutated α-crystallin, as Hsp16.5 with the analogous mutation shows conformational changes similar to those observed after substrate binding.

From this study, we have gained insight into the structure and mechanism of polydisperse human sHSPs. The structural analysis of Hsp16.5 variants suggests that sequence divergence in the N-terminal region leads to the wide spectrum of quaternary structures in the sHSP family. We propose that the dynamic and polydisperse nature of sHSPs is important for chaperone function.

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