Local environmental change from the G- to F-form of the actin molecule detected on anisotropy decay measurement

The fluorescence intensity has been reported to increase 10 to 25 times when N-(l-pyrene)-iodoacetamide (PIAA)-conjugated actin polymerizes from the G- to the F-form. To elucidate the molecular mechanism underlying this process, we measured the time-averaged aniso-tropy of PIAA-actin in both the G- and F-forms. The anisotropy ratio of PIAA-G-actin (0.137 ±0.008) was smaller than that of PIAA-F-actin (0.221 ±0.008). Similar results were obtained when N-(l-pyrene)-3-iodopropionamide (PIPA), a PIAA analogue with an extra carbon-chain in its reactive group, was conjugated with actin. The anisotropy ratio increased from 0.096±0.042 to 0.199±0.035 when PIPA-actin was transformed from the G-to the F-form. Further more, we measured the anisotropy decay of PIAA-actin in both the G- and F-forms. Least-square fitting revealed that the decay pattern was well fitted the wobbling-in-cone model. In the G-form, the pyrene of PIAA actin diffused in a cone region with a vertical half angle of 33.4'. This value decreased to 25.9• when the actin was transformed to the F-form, Because PIAA and PIPA were conjugated at Cys-374 of actin, our results suggest that a small cleft exists in the actin molecule in the vicinity of Cys-374, and this cleft becomes narrow upon polymerization, resulting in an increase in fluorescence intensity.