Dynamics of Bi-Functional Labeled Tropomyosin in Muscle Ghost Fiber Monitored by Saturation Transfer EPR

Abstract

Tropomyosin (Tm), an alpha-helical coiled-coil protein, is a key regulatory protein in muscle contraction. To date, little is known about the extent of Tm flexibility and the role of Tm dynamics in muscle regulation. In this work, the flexibility of two different regions of Tm was assessed using Saturation Transfer Electron Paramagnetic Resonance (ST EPR). In order to fully immobilize the spin probe on the surface of Tm we used a bi-functional spin label attached to i, i+4 positions of the coiled-coil obtained by cysteine mutagenesis. We have used conventional EPR and ST-EPR to detect wide range of dynamics from the very slow (millisecond) motions to fast sub-nanosecond modes. The labeled Tm mutants were reconstituted into “ghost muscle fibers” from which the myosin filaments and intrinsic regulatory proteins (tropomyosin, troponin) were removed.ST-EPR of the two mid-region mutants Tm H153C/D157C and Tm G188C/E192C as well as the C-terminus mutant Tm A268C/E272 gave a correlation time of 10.5 us ± 4.5 us, 42.5 us ± 27.5 us, and 42.5 us ± 27.5 us respectively (using H”/H and L”/L ratios of V'2 spectra). The difference in correlation time between the different di-mutants is an indication of the differential flexibility of the Tm protein. The study of the N-terminus (L13C/N17C) di-mutant will give us an additional understanding of Tm flexibility. Finally the introduction of Troponin complex (Tn) as well as S1 head of myosin under high and low calcium concentrations will give a complete picture of the dynamics of Tm in muscle regulation.