Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2013)

Heat Shock Protein 90 (Hsp90) is a highly conserved molecular chaperone necessary for eukaryotic life. This member of the cellular folding machinery has been extensively studied in recent years due to its intimate link to fundamental biological pathways that govern cellular homeostasis and disease.

Hsp90 undergoes large ATP dependant conformational changes that are necessary for client maturation in vivo. Though much progress had been made in building a model for a functional cycle of Hsp90, the fundamental question of how Hsp90 functions to re-model clients remained unclear.

Several forms of Hsp90 can be found throughout evolution with most higher eukaryotes having four homologs: two in the cytosol (Hsp90α, Hsp90β), one in the endoplasmic reticulum (GRP94) and one in the mitochondria (TRAP1).

Though not extensively studied at the time, I set out to investigate the structure and function of TRAP1, originally inspired by the balance between a higher eukaryotic Hsp90 and a simplified Hsp90 being most similar to the bacterial Hsp90 (bHsp90). With the latter comes the advantage of a more simple system where the large number of cochaperones identified for Hsp90α/β are not known to be required for client interactions with TRAP. Further, unique biology and disease links of TRAP1 were emerging with in vivo data showing that TRAP1 had unique ties to cellular homeostasis through interactions with client protein cyclophilin D (CypD), the master regulator of the mitochondrial permeability transition pore (mtPTP) and thus necrotic cell death.

Through this work I developed several biochemical/biophysical assays and strategies for working with TRAP1 in vitro, and through collaboration elucidated both shared and unique aspects of Hsp90 mechanism. Importantly, this work has provided a new hypothesis for Hsp90 mechanism of action with client proteins, which contributes a new model for the productive use of ATP hydrolysis for client re-modeling.

    Cover page: Structure and Function of the Mitochondrial Hsp90, TRAP1
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2014)
    Hsp90 is a conserved chaperone that facilitates protein homeostasis. Our crystal structure of the mitochondrial Hsp90, TRAP1, revealed an extension of the N-terminal β-strand previously shown to cross between protomers in the closed state. In this study, we address the regulatory function of this extension or 'strap' and demonstrate its responsibility for an unusual temperature dependence in ATPase rates. This dependence is a consequence of a thermally sensitive kinetic barrier between the apo 'open' and ATP-bound 'closed' conformations. The strap stabilizes the closed state through trans-protomer interactions. Displacement of cis-protomer contacts from the apo state is rate-limiting for closure and ATP hydrolysis. Strap release is coupled to rotation of the N-terminal domain and dynamics of the nucleotide binding pocket lid. The strap is conserved in higher eukaryotes but absent from yeast and prokaryotes suggesting its role as a thermal and kinetic regulator, adapting Hsp90s to the demands of unique cellular and organismal environments.
      Cover page: A novel N-terminal extension in mitochondrial TRAP1 serves as a thermal regulator of chaperone activity
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2014)
      Structural characterization of biologically important proteins faces many challenges associated with degradation of resolution as molecular size increases and loss of resolution improving tools such as perdeuteration when non-bacterial hosts must be used for expression. In these cases, sparse isotopic labeling (single or small subsets of amino acids) combined with long range paramagnetic constraints and improved computational modeling offer an alternative. This perspective provides a brief overview of this approach and two discussions of potential applications; one involving a very large system (an Hsp90 homolog) in which perdeuteration is possible and methyl-TROSY sequences can potentially be used to improve resolution, and one involving ligand placement in a glycosylated protein where resolution is achieved by single amino acid labeling (the sialyltransferase, ST6Gal1). This is not intended as a comprehensive review, but as a discussion of future prospects that promise impact on important questions in the structural biology area.
        Cover page: Sparse labeling of proteins: Structural characterization from long range constraints
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2014)
        While structural symmetry is a prevailing feature of homo-oligomeric proteins, asymmetry provides unique mechanistic opportunities. We present the crystal structure of full-length TRAP1, the mitochondrial Hsp90 molecular chaperone, in a catalytically active closed state. The TRAP1 homodimer adopts a distinct, asymmetric conformation, where one protomer is reconfigured via a helix swap at the middle:C-terminal domain (MD:CTD) interface. This interface plays a critical role in client binding. Solution methods validate the asymmetry and show extension to Hsp90 homologs. Point mutations that disrupt unique contacts at each MD:CTD interface reduce catalytic activity and substrate binding and demonstrate that each protomer needs access to both conformations. Crystallographic data on a dimeric NTD:MD fragment suggests that asymmetry arises from strain induced by simultaneous NTD and CTD dimerization. The observed asymmetry provides the potential for an additional step in the ATPase cycle, allowing sequential ATP hydrolysis steps to drive both client remodeling and client release.
          Cover page: Structural Asymmetry in the Closed State of Mitochondrial Hsp90 (TRAP1) Supports a Two-Step ATP Hydrolysis Mechanism
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2020)
          The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.
            Cover page: Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2020)
            The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.
              Cover page: Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.
              Top