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A Single-molecule Approach to Study Multimeric Molecular Motors and Optimal Thermodynamic Length

  • Author(s): Tafoya, Sara
  • Advisor(s): Bustamante, Carlos J.
  • et al.
Abstract

Single molecule techniques are uniquely informative for kinetic processes. As a result, in recent years they have become the methods of choice to interrogate many complex biomolecular systems (Bustamante & Tafoya 2017). During my PhD, I used optical tweezers, a technique for single-molecule manipulation, to study various biological processes.

First, I revisited the high internal pressure built inside the viral capsid of the bacteriophage phi29 during genome encapsidation (Liu et al. 2014b). During assembly of double-stranded DNA bacteriophages, the viral genome is encapsidated by a DNA packaging motor. High internal pressure builds up inside the viral capsid as a result of entropic and electrostatic repulsive forces resulting from DNA confinement. Previous single-molecule studies have determined the value of the internal pressure to be as high as 110 pN towards the end of DNA packaging. However, this value seemed overly high based on theoretical calculations. Using higher resolution data than in previous studies, my colleagues and I showed that the internal pressure reaches ~ 20 +/- 7 pN at 100% capsid filling, which is in better agreement with previous theoretical models.

Second, I determined the molecular mechanism for inter-subunit coordination in a viral ring ATPase. Subunits in multimeric ring motors must coordinate their enzymatic activity to perform their function (Tafoya et al. 2017). The bacteriophage phi29 DNA packaging motor is a pentameric ring ATPase whose subunits have been shown to operate in a highly coordinated manner. Therefore, this system is ideal to investigate how global subunit coordination can arise from stochastic processes and local molecular interactions. Using single-molecule optical tweezers and targeted mutagenesis, I showed that coordination arises from inter-subunit enzymatic regulation.The subunits use their arginine finger to promote nucleotide exchange and to activate ATP hydrolysis in their neighbors. These regulatory processes display similar features to those observed in small GTPases.

Third, in light of what I learned about the phi29 DNA packaging motor's operation, I reviewed various mechanisms of small GTPase-like regulation in different motor proteins (Tafoya & Bustamante 2017). In particular, I highlighted the fact that all these mechanisms share a general feature: the motor's function is controlled by stimulation or repression of its ATPase activity, which is regulated allosterically by different factors.

Finally, I tested a prediction from fluctuation theorems to minimize the thermodynamic length in a process out of equilibrium (Tafoya et al. 2017b). Genome encapsidation by the phi29 DNA packaging motor is only an example of the multiple non-equilibrium processes occurring in the cell. In fact, to maintain their organization, biological systems must operate far from equilibrium, continuously utilizing and dissipating energy. Non-equilibrium theory is underdeveloped, but recent work has approximated the excess work in processes out of equilibrium. I tested this theory's predictions performing pulling experiments on a DNA hairpin. I found that the predicted minimum-dissipation protocols indeed require significantly less work than naive ones across a wide span of driving velocities.

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