Allosteric Communication and Force Generation in Myosin Motors

Open Access
Author:
Trivedi, Darshan V
Graduate Program:
Physiology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
September 29, 2014
Committee Members:
  • Christopher Martin Yengo, Dissertation Advisor
  • Christopher Martin Yengo, Committee Chair
  • Lisa M Shantz, Committee Member
  • Thomas E Spratt, Special Member
  • Blaise Peterson, Committee Member
  • William O Hancock, Committee Member
Keywords:
  • Myosin
  • Actin
  • FRET
  • Motor Proteins
Abstract:
Muscle contraction, intracellular transport and a myriad of other mechanical functions in a cell are governed by myosin motors. Myosins utilize the chemical energy derived from ATP hydrolysis to perform mechanical work via a cyclic interaction with actin filaments. Intricate allosteric pathways operate within the myosin molecule which leads to the coupling of different sub-domains and an efficient generation of force. The three main regions involved in this active communication are the nucleotide and actin-binding regions which are both coupled to the force-generating lever arm region. The lever arm undergoes a reversible movement defined by the recovery stroke and the powerstroke which eventually leads to the generation of force. However, the kinetic and structural details of this mechanism of force generation remain elusive. At an interface of biochemistry and biophysics, this study utilizes novel fluorophore labeling strategies combined with fluorescence spectroscopy and stopped-flow kinetics to answer these questions. Myosin V (MV) is used as a model to uncover the structural changes associated with the catalytic cycle. The rate-limiting conformational change of MV is a closed-to-open transition of the nucleotide binding region prior to the release of ADP. This study investigates the role of a specific structural element called as switch II and the magnesium (Mg) ion in the coupling between the nucleotide-and actin binding regions and their role in mediating the rate-limiting transition. Switch II was found to be critical in both these processes, while Mg played a central role in modulating the rate-limiting transition prior to ADP release. A long standing question about the precise temporal kinetics of the lever arm swing in relation to the product release steps and force generation is also unambiguously answered by this work. A novel fluorophore labeling strategy combined with stopped-flow FRET experiments unravels the kinetic mechanism of lever arm swing. The recovery stroke occurs concurrent with formation of the hydrolysis competent state. The powerstroke occurs in two phases, a fast phase precedes phosphate release and a slow phase precedes the release of ADP. These results provide direct evidence for the order of events associated with force generation in myosins.