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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2018)

Proteins are one of the main building blocks of life. Among their numerous functions are roles as biocatalysts for essential chemical reactions, signaling agents that coordinate communication down to the sub-cellular level, and as modules for constructing cellular superstructures. In many cases, these functions rely on the ability of proteins to form stable and specific associative interactions with other proteins, as well as with metal ions. Drawing inspiration from nature, we deploy these functional features as design elements that aid in the construction of artificial metalloprotein assemblies. Using an iterative design approach, we have engineered the monomeric protein cytochrome cb562 by application orthogonal strategies: Metal Designed Protein Self-Assembly (MDPSA), Metal-Templated Interface Redesign (MeTIR), and the installation of intermolecular disulfide bond crosslinks. This synergistic design approach afforded a self-assembling protein variant, C38/C81/C96R1, which bears metal chelating groups, a designed dimerization interface (termed i1), and surface-exposed cysteine residues. In the presence of ZnII this engineered variant self-assembled into a tetramer, Zn-C38/C81/C96R14. Removal of ZnII from the assembled tetramer resulted in hydrolysis of a single C38-C38 disulfide bond, leaving the five remaining crosslinks intact. Thus, C38/C81/C96R14 is an allosteric protein, capable of remotely coupling ZnII binding at is central binding sites to the breakage of a peripheral disulfide bond. In this work, we discuss the design of the C38/C81/C96R14 protein as well as the demonstration of this allosteric behavior. We further carry out biochemical and biophysical characterization of this protein and related variants to determine the structural and energetic basis of this Zn-disulfide allostery. The C38-C38 disulfide bonds are embedded in the i1interface which, crucially, forms malleable protein-protein contacts. The flanking disulfide crosslinks of C38/C81/C96R14 serve as structural conduits for coupling the pair of i1 interfaces, which is critical to develop the requisite driving force to effect disulfide bond hydrolysis. In this designed system, it is the underlying malleability of the structure that gives rise to the allosteric behavior. We consider this as a successful application of synergistic design to give a coordinated protein function, and anticipate that adoption of similar design paradigms will greatly benefit ongoing protein engineering efforts in the community at large.

    Cover page: Design and Characterization of an Allosteric Metalloprotein Assembly
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2011)
    Potassium currents generated by voltage-gated potassium (Kv) channels comprising α-subunits from the Kv1, 2, and 3 subfamilies facilitate high-frequency firing of mammalian neurons. Within these subfamilies, only three α-subunits (Kv1.4, Kv3.3, and Kv3.4) generate currents that decay rapidly in the open state because an N-terminal ball domain blocks the channel pore after activation-a process termed N-type inactivation. Despite its importance to shaping cellular excitability, little is known of the processes regulating surface expression of N-type α-subunits, versus their slowly inactivating (delayed rectifier) counterparts. Here we found that currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels are all strongly suppressed by the single transmembrane domain ancillary (β) subunits KCNE1 and KCNE2. A combination of electrophysiological, biochemical, and immunofluorescence analyses revealed this suppression is due to KCNE1 and KCNE2 retaining Kv1.4 and Kv3.4 intracellularly, early in the secretory pathway. The retention is specific, requires α-β coassembly, and does not involve the dynamin-dependent endocytosis pathway. However, the small fraction of Kv3.4 that escapes KCNE-dependent retention is regulated by dynamin-dependent endocytosis. The findings illustrate two contrasting mechanisms controlling surface expression of N-type Kv α-subunits and therefore, potentially, cellular excitability and refractory periods.
      Cover page: KCNE1 and KCNE2 Inhibit Forward Trafficking of Homomeric N-Type Voltage-Gated Potassium ChannelsCreative Commons 'BY' version 4.0 license
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2011)
      Voltage-gated potassium (Kv) currents generated by N-type α-subunit homotetramers inactivate rapidly because an N-terminal ball domain blocks the channel pore after activation. Hence, the inactivation rate of heterotetrameric channels comprising both N-type and non-N-type (delayed rectifier) α-subunits depends upon the number of N-type α-subunits in the complex. As Kv channel inactivation and inactivation recovery rates regulate cellular excitability, the composition and expression of these heterotetrameric complexes are expected to be tightly regulated. In a companion article, we showed that the single transmembrane segment ancillary (β) subunits KCNE1 and KCNE2 suppress currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels, by trapping them early in the secretory pathway. Here, we show that this trapping is prevented by coassembly of the N-type α-subunits with intra-subfamily delayed rectifier α-subunits. Extra-subfamily delayed rectifier α-subunits, regardless of their capacity to interact with KCNE1 and KCNE2, cannot rescue Kv1.4 or Kv3.4 surface expression unless engineered to interact with them using N-terminal A and B domain swapping. The KCNE1/2-enforced checkpoint ensures N-type α-subunits only reach the cell surface as part of intra-subfamily mixed-α complexes, thereby governing channel composition, inactivation rate, and-by extension-cellular excitability.
        Cover page: KCNE1 and KCNE2 Provide a Checkpoint Governing Voltage-Gated Potassium Channel α-Subunit CompositionCreative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2020)
        TOKs are outwardly rectifying K+ channels in fungi with two pore-loops and eight transmembrane spans. Here, we describe the TOKs from four pathogens that cause the majority of life-threatening fungal infections in humans. These TOKs pass large currents only in the outward direction like the canonical isolate from Saccharomyces cerevisiae (ScTOK), and distinct from other K+ channels. ScTOK, AfTOK1 (Aspergillus fumigatus), and H99TOK (Cryptococcus neoformans grubii) are K+ -selective and pass current above the K+ reversal potential. CaTOK (Candida albicans) and CnTOK (Cryptococcus neoformans neoformans) pass both K+ and Na+ and conduct above a reversal potential reflecting the mixed permeability of their selectivity filter. Mutations in CaTOK and ScTOK at sites homologous to those that open the internal gates in classical K+ channels are shown to produce inward TOK currents. A favored model for outward rectification is proposed whereby the reversal potential determines ion occupancy, and thus, conductivity, of the selectivity filter gate that is coupled to an imperfectly restrictive internal gate, permitting the filter to sample ion concentrations on both sides of the membrane.
          Cover page: TOK channels use the two gates in classical K+ channels to achieve outward rectificationCreative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2020)
          TOKs are outwardly rectifying K+ channels in fungi with two pore-loops and eight transmembrane spans. Here, we describe the TOKs from four pathogens that cause the majority of life-threatening fungal infections in humans. These TOKs pass large currents only in the outward direction like the canonical isolate from Saccharomyces cerevisiae (ScTOK), and distinct from other K+ channels. ScTOK, AfTOK1 (Aspergillus fumigatus), and H99TOK (Cryptococcus neoformans grubii) are K+ -selective and pass current above the K+ reversal potential. CaTOK (Candida albicans) and CnTOK (Cryptococcus neoformans neoformans) pass both K+ and Na+ and conduct above a reversal potential reflecting the mixed permeability of their selectivity filter. Mutations in CaTOK and ScTOK at sites homologous to those that open the internal gates in classical K+ channels are shown to produce inward TOK currents. A favored model for outward rectification is proposed whereby the reversal potential determines ion occupancy, and thus, conductivity, of the selectivity filter gate that is coupled to an imperfectly restrictive internal gate, permitting the filter to sample ion concentrations on both sides of the membrane.
            Cover page: TOK channels use the two gates in classical K+ channels to achieve outward rectification.
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