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-(1-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 anisotropy 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-(1-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 degrees. This value decreased to 25.9 degrees 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.
Sasaki
Y
Y
Laboratory of Chemistry, Faculty of General Education, Tokyo University of Agriculture and Technology.
Tsunomori
F
F
Yamashita
T
T
Horie
K
K
Ushiki
H
H
Ishikawa
R
R
Kohama
K
K
D000199:Actins / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007460:Iodoacetamide / Q000031:analogs & derivatives; D011487:Protein Conformation
D000199:Actins; D005456:Fluorescent Dyes; C029358:N-(1-pyrenyl)iodoacetamide; D003545:Cysteine; D007460:Iodoacetamide