Muscle contraction is driven by the actin-activated hydrolysis of ATP by myosin, resulting
in the relative sliding of actin and myosin filaments. Current models propose that filament
sliding is driven by a structural transition of myosin’s catalytic domain (CD) and light
chain domain (LCD). The goal of this research is to measure structural transitions of
myosin II (muscle and nonmuscle) that are associated for force generation. Structural
measurements were made using electron paramagnetic resonance (EPR) spectroscopy. This
work is comprised of two separate, but related, projects.
In the first project (Chapter 3), thiol crosslinking and EPR were used to resolve
structural transitions of myosin’s LCD and CD that are associated with force generation.
Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled
regulatory light chain (RLC) for endogenous RLC, with full retention of function. LCD
orientation and dynamics were measured in three biochemical states: relaxation (A.M.T),
post-hydrolysis intermediate (A.M.D.P), and rigor (A.M.D). To trap myosin in a
structural state analogous to the elusive post-hydrolysis ternary complex A.M.D.P, we
used pPDM to crosslink SH1 (Cys707) to SH2 (Cys697) on the CD. EPR showed that the LCD of crosslinked fibers has an orientational distribution intermediate between
relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics
indistinguishable from that of rigor. Similar results were obtained for the CD using a
bifunctional spin label to crosslink SH1 to SH2, but the CD was more disordered than the
LCD. We conclude that SH1-SH2 crosslinking traps a state in which both the LCD and
CD are in a structural state intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis
that the crosslinked state is an A.M.D.P analog on the force generation pathway.
In the second project, we present a method for obtaining high-resolution structural
information of proteins using EPR of a bifunctional spin label (BSL). Two
complimentary EPR techniques were employed to measure dynamics and orientation
(conventional EPR) and intraprotein distances (dipolar electron-electron resonance). The
exploitation of BSL is a key feature of this work. BSL attaches at residue positions i and
i+4, which drastically restricts probe motion compared to monofunctional probes. For
comparison, measurements were also made with the monofunctional spin label MSL.
Subfragment 1 of Dictyostelium myosin II (S1dC) was used to exemplify the increased resolution provided by BSL. Using this approach, we demonstrate with experiments that
BSL significantly increases resolution when measuring distance and orientation
compared to MSL. And while this work does focus on the methodology, there is
significant biological insight into myosin’s nucleotide-dependent structural transitions.
University of Minnesota Ph.D. dissertation. June 2012. Major: Biophysical Sciences and Medical Physics. Advisor: David D. Thomas. 1 computer file (PDF); x, 99 pages.
Mello, Ryan Nicholas.
Structural transitions of myosin associated with force generation in spin-labeled muscle fibers..
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