Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. III. Changes in the conformation of tropomyosin associated with functional states.
The local conformational changes in the tropomyosin molecule under various conditions were studied by means of fluorimetry using SH-directed fluorescent dyes, N-(1-anilinonaphthyl-4)maleimide (ANM) and N-(3-pyrene)maleimide (PRM). 1. The fluorescence intensity, polarization and the emmission maximum of ANM-tropomyosin were found to be susceptible to ionic strength, but in different ways. The changes in these parameters suggest that the fluorescence-labeled sulfhydryl group or groups become more buried in a hydrophobic internal region by salt-induced depolymerization of aggregate and by adding F-actin to tropomyosin. 2. Titration of the labeled tropomyosin with F-actin revealed a cooperative nature in ANM labeling and a simple saturation kinetics in PRM labeling. The dissociation constant of F-actin to PRM-tropomyosin was calculated to be 5.8-10(-6) M. 3. Temperature dependence of the fluorescence polarization showed a thermal transition in the conformation of ANM- or PRM-tropomyosin at around 30 degrees C. Flexibility or segmental motion of the region containing the fluorophore was suppressed significantly on adding troponin and markedly on adding F-actin. 4. Measurements of the quantum yield and polarization of the ANM-tropomyosin-F-actin complex suggested that troponin strengthened the binding between the two proteins and that Ca2+ reversed this effect.
D000199:Actins; D001665:Binding Sites; D002118:Calcium; D046911:Macromolecular Substances; D008301:Maleimides; D009124:Muscle Proteins; D009281:Naphthalenes; D011485:Protein Binding; D011487:Protein Conformation; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin; D014336:Troponin
D000199:Actins; D046911:Macromolecular Substances; D008301:Maleimides; D009124:Muscle Proteins; D009281:Naphthalenes; D014335:Tropomyosin; D014336:Troponin; D002118:Calcium