Conformational changes in bovine heart myosin as studied by EPR and DSC techniques.
Thermal behavior of intact and LC-2 deficient myosin obtained from bovine heart was studied using EPR and DSC techniques. The reactive thiol sites (Cys 704) of myosin was labelled with 4-maleimidopiperidine-nitroxyl, and the measurements were taken in X-band in the conventional and saturation transfer EPR time domains. DSC scans were made from 5 degrees up to 60 degrees C with 0.25 degree C/min scan rate. Bovine heart myosin was isolated by standard methods. The LC-2 deficient myosin was prepared by cleaving myosin with alpha-chymotrypsin (400:1 molar ratio) for 1.5 min at 25 degrees. Our basic finding was a conformational change in LC-2 deficient myosin detected at 18 degrees C. It was not observed in intact myosin suggesting that the dissociation of the regulatory light chain resulted in a local structural change in the neighbourhood of the attached label in the 20 kD domain. The rotational correlation time of the label and the microwave saturation behavior of myosin at 25 degrees C exhibited no significant differences after removal of the LC-2 light chain. However, the mobility of the same label was significantly diminished in skeletal muscle. Studying the melting behavior of myosin, six endothermic peaks were detected at 19; 41.3; 43.3; 45.5; 48.5; and 54.3 degrees C (enthalpies: 708.4; 399; 773.8; 1089; 1612.8; and 3304.8 kJ/mol). They were assigned to the segment containing the essential thiols: HMM S-2, HMM S-1 (50kD and 20kD plus 27kD) and LMM. Removal of the LC-2 light chain was associated with the disappearance of the 18 degrees transition showing again a structural change in LC-2 deficient myosin which extended to a larger region.
Lörinczi
D
D
Research Group of Hungarian Academy of Sciences in Biophysical Institute, University Medical School, Pécs.
Hoffmann
U
U
Pótó
L
L
Belágyi
J
J
Laggner
P
P
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D066298:In Vitro Techniques; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013113:Spin Labels; D013816:Thermodynamics
D013113:Spin Labels; D000244:Adenosine Diphosphate; D009218:Myosins