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

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2017)

The viral DNA packaging motors are among the most powerful biological machines known, and their mechanisms have been of interest for decades. The work presented in this dissertation has been dedicated to studying these mechanisms on the example of the T4 and Lambda bacteriophages, which are well known model organisms.

Custom-built optical tweezers were utilized for real time observations of single DNA molecule packaging. A force-feedback loop allowed us to study the DNA translocation fueled by ATP hydrolysis under different conditions.

The regulation of the motors grip on the DNA by the nucleotide state was investigated through a novel technique, and the interaction of the motor subunits with the DNA was shown to depend on the state of the ATP-binding pockets. This finding brings us closer to understanding the level of coordination between the subunits during translocation. The fact that the motors grip on the DNA depends on the nucleotide state also allows to judge about the state of the ATP-binding pocket based on the packaging dynamics. A novel mechanism of the clamping of the DNA by the packaging complex to prevent dissociation was identified in the course of these experiments.

The role of the putative conserved Walker A and Walker B ATP binding motifs were examined through mutagenesis studies. An extensive study of 23 different mutants in single-molecule experiments was conducted, and it was concluded that these regions are indeed involved in ATP-binding, but we also identified residues from this region that are important for ATP-alignment, catalysis of hydrolysis, and mechanochemical coupling. In addition measurements on the mutation of the glutamate residue E179 directly downstream of Walker B were performed, and it confirmed that this residue catalyzes the hydrolysis.

A conserved Loop-Helix-Loop region previously studied in this lab, and proposed to play a regulatory role in translocation velocity was also studied. 8 different mutants were probed in the single-molecule assay, and, where packaging was detected, the effect on the translocation speed was studied.

This is, to date, the most extensive mutagenesis study preformed to investigate the roles of particular amino acids in the detailed function of a viral packaging motor..

    Cover page: Studies of viral DNA packaging motor mechanism on a single molecule level via optical tweezers
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2020)
    Calmodulin-dependent kinase II (CaMKII) has long been known to play an important role in learning and memory as well as long term potentiation (LTP). More recently it has been suggested that it might be involved in the time averaging of synaptic signals, which can then lead to the high precision of information stored at a single synapse. However, the role of the scaffolding molecule, neurogranin (Ng), in governing the dynamics of CaMKII is not yet fully understood. In this work, we adopt a rule-based modeling approach through the Monte Carlo method to study the effect of Ca2+ signals on the dynamics of CaMKII phosphorylation in the postsynaptic density (PSD). Calcium surges are observed in synaptic spines during an EPSP and back-propagating action potential due to the opening of NMDA receptors and voltage dependent calcium channels. Using agent-based models, we computationally investigate the dynamics of phosphorylation of CaMKII monomers and dodecameric holoenzymes. The scaffolding molecule, Ng, when present in significant concentration, limits the availability of free calmodulin (CaM), the protein which activates CaMKII in the presence of calcium. We show that Ng plays an important modulatory role in CaMKII phosphorylation following a surge of high calcium concentration. We find a non-intuitive dependence of this effect on CaM concentration that results from the different affinities of CaM for CaMKII depending on the number of calcium ions bound to the former. It has been shown previously that in the absence of phosphatase, CaMKII monomers integrate over Ca2+ signals of certain frequencies through autophosphorylation (Pepke et al, Plos Comp. Bio., 2010). We also study the effect of multiple calcium spikes on CaMKII holoenzyme autophosphorylation, and show that in the presence of phosphatase, CaMKII behaves as a leaky integrator of calcium signals, a result that has been recently observed in vivo. Our models predict that the parameters of this leaky integrator are finely tuned through the interactions of Ng, CaM, CaMKII, and PP1, providing a mechanism to precisely control the sensitivity of synapses to calcium signals. Author Summary not valid for PLOS ONE submissions.
      Cover page: Interactions between calmodulin and neurogranin govern the dynamics of CaMKII as a leaky integrator
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2018)
      ATP-powered viral packaging motors are among the most powerful biomotors known. Motor subunits arranged in a ring repeatedly grip and translocate the DNA to package viral genomes into capsids. Here, we use single DNA manipulation and rapid solution exchange to quantify how nucleotide binding regulates interactions between the bacteriophage T4 motor and DNA substrate. With no nucleotides, there is virtually no gripping and rapid slipping occurs with only minimal friction resisting. In contrast, binding of an ATP analog engages nearly continuous gripping. Occasional slips occur due to dissociation of the analog from a gripping motor subunit, or force-induced rupture of grip, but multiple other analog-bound subunits exert high friction that limits slipping. ADP induces comparably infrequent gripping and variable friction. Independent of nucleotides, slipping arrests when the end of the DNA is about to exit the capsid. This end-clamp mechanism increases the efficiency of packaging by making it essentially irreversible.
        Cover page: Nucleotide-dependent DNA gripping and an end-clamp mechanism regulate the bacteriophage T4 viral packaging motor
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2016)
        During the assembly of many viruses, a powerful ATP-driven motor translocates DNA into a preformed procapsid. A Walker-A "P-loop" motif is proposed to coordinate ATP binding and hydrolysis with DNA translocation. We use genetic, biochemical, and biophysical techniques to survey the roles of P-loop residues in bacteriophage lambda motor function. We identify 55 point mutations that reduce virus yield to below detectable levels in a highly sensitive genetic complementation assay and 33 that cause varying reductions in yield. Most changes in the predicted conserved residues K76, R79, G81, and S83 produce no detectable yield. Biochemical analyses show that R79A and S83A mutant proteins fold, assemble, and display genome maturation activity similar to wild-type (WT) but exhibit little ATPase or DNA packaging activity. Kinetic DNA cleavage and ATPase measurements implicate R79 in motor ring assembly on DNA, supporting recent structural models that locate the P-loop at the interface between motor subunits. Single-molecule measurements detect no translocation for K76A and K76R, while G81A and S83A exhibit strong impairments, consistent with their predicted roles in ATP binding. We identify eight residue changes spanning A78-K84 that yield impaired translocation phenotypes and show that Walker-A residues play important roles in determining motor velocity, pausing, and processivity. The efficiency of initiation of packaging correlates strongly with motor velocity. Frequent pausing and slipping caused by changes A78V and R79K suggest that these residues are important for ATP alignment and coupling of ATP binding to DNA gripping. Our findings support recent structural models implicating the P-loop arginine in ATP hydrolysis and mechanochemical coupling.
          Cover page: Walker-A Motif Acts to Coordinate ATP Hydrolysis with Motor Output in Viral DNA Packaging
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2019)
          Many viruses employ ATP-powered motors for genome packaging. We combined genetic, biochemical, and single-molecule techniques to confirm the predicted Walker-B ATP-binding motif in the phage λ motor and to investigate the roles of the conserved residues. Most changes of the conserved hydrophobic residues resulted in >107-fold decrease in phage yield, but we identified nine mutants with partial activity. Several were cold-sensitive, suggesting that mobility of the residues is important. Single-molecule measurements showed that the partially active A175L exhibits a small reduction in motor velocity and increase in slipping, consistent with a slowed ATP binding transition, whereas G176S exhibits decreased slipping, consistent with an accelerated transition. All changes to the conserved D178, predicted to coordinate Mg2+•ATP, were lethal except conservative change D178E. Biochemical interrogation of the inactive D178N protein found no folding or assembly defects and near-normal endonuclease activity, but a ∼200-fold reduction in steady-state ATPase activity, a lag in the single-turnover ATPase time course, and no DNA packaging, consistent with a critical role in ATP-coupled DNA translocation. Molecular dynamics simulations of related enzymes suggest that the aspartate plays an important role in enhancing the catalytic activity of the motor by bridging the Walker motifs and precisely contributing its charged group to help polarize the bound nucleotide. Supporting this prediction, single-molecule measurements revealed that change D178E reduces motor velocity without increasing slipping, consistent with a slowed hydrolysis step. Our studies thus illuminate the mechanistic roles of Walker-B residues in ATP binding, hydrolysis, and DNA translocation by this powerful motor.
            Cover page: Functional Dissection of a Viral DNA Packaging Machine's Walker B Motif
            Top