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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2016)

Membrane proteins are a neglected, but important class of proteins throughout the biological world. They carry out critical roles in the cell due to their unique location, such as transport across a membrane, transduction of exterior signals, and interaction between discrete aqueous regions. Despite the importance of these proteins, understanding of how they fold has lagged far behind that of soluble proteins. One of the primary challenges to studying membrane protein folding is developing methods that interrogate folding in the native environment of the lipid bilayer. Our lab has developed a method for measuring membrane protein stability under native conditions using a secondary protein that preferentially binds the unfolded state, obviating the need for harsh denaturants. Employing this method with a multimeric polytopic membrane protein, we measured an extremely slow unfolding rate, demonstrating that α-helical membrane proteins can have high kinetic stability under non-denaturing conditions. Efforts were made to expand the steric trap method for single-molecule fluorescence measurements in lipid vesicles, but were ultimately stymied by the inability to preserve the trapped complexes for measurement. Our lab has also applied single-molecule techniques to membrane protein folding. We were able to map the energy landscape of a membrane protein in a lipid bilayer using forced unfolding driven by magnetic tweezers. Further advancements to this technique simplified the attachment chemistry to ready the protein for tweezing. These techniques can be applied to a wide array of membrane proteins in a broad spectrum of membrane environments.

    Cover page: Uncovering Membrane Protein Stability Under Native Conditions
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2016)
    Manipulating single molecules and systems of molecules with mechanical force is a powerful technique to examine their physical properties. Applying force requires attachment of the target molecule to larger objects using some sort of molecular tether, such as a strand of DNA. DNA handle attachment often requires difficult manipulations of the target molecule, which can preclude attachment to unstable, hard to obtain, and/or large, complex targets. Here we describe a method for covalent DNA handle attachment to proteins that simply requires the addition of a preprepared reagent to the protein and a short incubation. The handle attachment method developed here provides a facile approach for studying the biomechanics of biological systems.
      Cover page: A simple DNA handle attachment method for single molecule mechanical manipulation experiments
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2013)
      Approximately 10% of water-soluble proteins are considered kinetically stable with unfolding half-lives in the range of weeks to millenia. These proteins only rarely sample the unfolded state and may never unfold on their respective biological time scales. It is still not known whether membrane proteins can be kinetically stable, however. Here we examine the subunit dissociation rate of the trimeric membrane enzyme, diacylglycerol kinase, from Escherichia coli as a proxy for complete unfolding. We find that dissociation occurs with a half-life of at least several weeks, demonstrating that membrane proteins can remain locked in a folded state for long periods of time. These results reveal that evolution can use kinetic stability to regulate the biological function of membrane proteins, as it can for soluble proteins. Moreover, it appears that the generation of kinetic stability could be a viable target for membrane protein engineering efforts.
        Cover page: Membrane Proteins Can Have High Kinetic Stability
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2018)
        Protein folding is a fundamental life process with many implications throughout biology and medicine. Consequently, there have been enormous efforts to understand how proteins fold. Almost all of this effort has focused on water-soluble proteins, however, leaving membrane proteins largely wandering in the wilderness. The neglect has occurred not because membrane proteins are unimportant but rather because they present many theoretical and technical complications. Indeed, quantitative membrane protein folding studies are generally restricted to a handful of well-behaved proteins. Single-molecule methods may greatly alter this picture, however, because the ability to work at or near infinite dilution removes aggregation problems, one of the main technical challenges of membrane protein folding studies.
          Cover page: Applications of Single-Molecule Methods to Membrane Protein Folding Studies
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2015)
          Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single-molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, Escherichia coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (ΔG = 6.5 kBT) but a large unfolding barrier (21.3 kBT) that can maintain the protein in a folded state for long periods of time (t1/2 ∼3.5 h). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure.
            Cover page: Mapping the energy landscape for second-stage folding of a single membrane protein
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2018)
            ClC chloride channels and transporters are important for chloride homeostasis in species from bacteria to human. Mutations in ClC proteins cause genetically inherited diseases, some of which are likely to involve folding defects. The ClC proteins present a challenging and unusual biological folding problem because they are large membrane proteins possessing a complex architecture, with many reentrant helices that go only partway through membrane and loop back out. Here we were able to examine the unfolding of the Escherichia coli ClC transporter, ClC-ec1, using single-molecule forced unfolding methods. We found that the protein could be separated into two stable halves that unfolded independently. The independence of the two domains is consistent with an evolutionary model in which the two halves arose from independently folding subunits that later fused together. Maintaining smaller folding domains of lesser complexity within large membrane proteins may be an advantageous strategy to avoid misfolding traps.
              Cover page: Unfolding of a ClC chloride transporter retains memory of its evolutionary history.
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