University of Minnesota
School of Physics & Astronomy

Biophysics Seminar

Thursday, March 22nd 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Yahor Savich (David Thomas lab), School of Physics and Astronomy, University of Minnesota
Subject: Myosin Orientation in a Functioning Muscle Fiber With High Angular Resolution

We have measured the orientation of myosin in a muscle fiber bundle using electron paramagnetic resonance (EPR) and a bifunctional spin label (BSL), with angular resolution of a few degrees. Despite advances in cryo-EM, fluorescence, and small-angle X-ray diffraction, these techniques do not provide high-resolution structural information about myosin heads in vitro under functional conditions. A pair of (i,i+4) Cys residues were engineered on an alpha-helix in the regulatory light chain (RLC). By exchanging endogenous RLC with BSL-labeled RLC on oriented muscle fibers, we were able to resolve angular distributions in several biochemical states due to the stereospecific attachment of BSL’s two disulfide bonds. In this setup, the accurate determination of BSL’s angular coordinates allowed us to determine the orientation of individual structural elements with respect to the muscle fiber axis. Addition of ATP in the absence of Ca, relaxing the muscle, shifted the orientational distribution to a much more disordered distribution. This work is inspired by growing therapeutic interest in super-relaxed myosin state, which predicts presence of order in relaxation.

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