Transient electrical birefringence characterization of heavy meromyosin.
Heavy meromyosin (HMM) and myosin subfragment 1 (S1) were prepared from myosin by using low concentrations of alpha-chymotrypsin. The light chain distribution in HMM was identical with that of myosin, within experimental error, when analyzed on 12% polyacrylamide gels after electrophoresis. Specific birefringences and birefringence decay times were measured by transient electrical birefringence in 5 mM KCl, 5 mM tris(hydroxymethyl)aminomethane (pH 7), and 1 mM MgCl2 at 4 degrees C under gentle conditions that reduced the CaATPase activity by less than 10%. For solutions of HMM, by use of electric field pulses shorter than 0.5 microseconds, the birefringence decay signal from the S1 portions of HMM could be resolved and the rotational motions of the S1 moieties observed directly. The rotation relaxation time, adjusted to 20 degrees C, was 0.34 microseconds; this is in quantitative agreement with previous hydrodynamic results obtained by using covalently attached probes. The assignment of the fast decay time obtained with HMM to the S1 portions was confirmed by birefringence decay measurements on free S1, for which the relaxation time was 0.13 microseconds, corrected to 20 degrees C. The specific birefringences for S1 and HMM, respectively, were 0.37 X 10(-6) and 12.8 X 10(-6) (cm/statvolt)2. Thus, for much longer electric field pulses, the signal from HMM is due almost entirely to its subfragment 2 (S2) portion, and its rotational dynamics can also be monitored directly by using electrical birefringence. The decay of the signal from the S2 portion could be adequately fit without evoking bending of the S2 portion of HMM other than at its junction with S1.
D001718:Birefringence; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation
D015879:Myosin Subfragments; D010446:Peptide Fragments; D002918:Chymotrypsin; D009218:Myosins