Analysis of nucleotide myosin complexes in skeletal muscle fibres by DSC and EPR.
The internal dynamics and thermal unfolding of fibre bundles prepared from rabbit psoas muscle has been studied in the presence of nucleotides by differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) spectroscopy. Using ADP, adenosine 5'-triphosphate (ATP), AMP.PNP and inorganic phosphate analogue orthovanadate (V(i)), AlF(4)(-) and BeF(3)(-), three intermediate states of the ATP hydrolysis cycle were simulated in glycerinated muscle fibres. In the main transition of the DSC pattern, three overlapping endotherms were detected in rigor, four in strongly as well as weakly binding state of myosin to actin. Deconvolution procedure showed that the transition temperature of 67.5 degrees C was the same for rigor and strong binding state of myosin. In contrast, nucleotide binding induced shift of the melting temperatures of 52 degrees C and 67.5 degrees C, appeared a new fourth peak at 74 and 77 degrees C and produced changes in the calorimetric enthalpies. The changes of the parameters of the peak functions suggest global rearrangements of the internal structure in myosin heads in the intermediate states. In the presence of ADP or ATP plus phosphate analogue orthovanadate or beryllium fluoride, aluminium fluoride, the conventional EPR spectra of spin-labeled muscle fibres showed large changes in the ordering of the probe molecules, and a new distribution of spin labels appeared. ATP plus orthovanadate induced the orientation disorder of myosin heads; the random population of spin labels gave evidence of large local conformational and motional changes in the internal structure of myosin heads. Saturation transfer EPR measurements reported increased rotational mobility of spin labels in the presence of ATP plus phosphate analogues corresponding to weakly binding state of myosin to actin.
Lorinczy
D
D
Biophysical Department, Faculty of Medicine, University of Pécs, H-7624, Pécs, 12 Szigeti Str, Hungary. denes.lorinczy@aok.pte.edu
Hartvig
N
N
Belagyi
J
J
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D017607:Aluminum Compounds; D000818:Animals; D002152:Calorimetry, Differential Scanning / Q000379:methods*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005459:Fluorides; D006358:Hot Temperature; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism; D015336:Molecular Probe Techniques; D018485:Muscle Fibers, Skeletal / Q000737:chemistry* / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D010758:Phosphorus / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D016658:Psoas Muscles; D051381:Rats; D013696:Temperature; D014638:Vanadates
D000199:Actins; D017607:Aluminum Compounds; D046911:Macromolecular Substances; D020409:Molecular Motor Proteins; D009711:Nucleotides; D010758:Phosphorus; D014638:Vanadates; D009218:Myosins; D005459:Fluorides; C032311:aluminum fluoride