DOMAIN MOTION IN ACTIN OBSERVED BY FLUORESCENCE RESONANCE ENERGY-TRANSFER

Actin is composed of two well-separated globular domains which are fur ther subdivided into two subdomains [Kabsch, W., Mannherz, H, G., Suck , D., Pal, E. F., and Holmes, K. C. (1990) Nature 347, 37-44]. Subdoma ins 1 and 2 constitute the small domain, and subdomains 3 and 4 compri se the large domain. In order to test a hinge bending domain motion in actin such as observed in many kinases, fluorescence resonance energy transfer between two probes attached to each of the two domains was m easured by steady-state and time-resolved fluorometers. The adenine ba se is bound in a hydrophobic pocket between subdomains 3 and 4, and Ty r-69 is located at subdomain 2. In the present study, the adenine moie ty was labeled with a fluorescence donor, epsilon ATP, and tyrosine-69 was labeled with the energy acceptor, dansyl chloride. Assuming the r andom orientation factor kappa(2) = 2/3, the distance between E-adenin e moiety and dansyl chloride attached to Tyr-69 in G-actin was determi ned to be 2.46 nm from steady-state fluorescence measurements. The add ition of DNase I did not appreciably change the distance (less than 0. 1 nm). The distance decreased to 2.27 nm during polymerization by the addition of phalloidin under physiological salt conditions. On the oth er hand, time-resolved fluorescence energy transfer measurements have been used to investigate a distribution of distances for a donor-accep tor pair. In G-actin, the mean distance between probes was 2.79 nm wit h a full width at half-maximum of 3.91 nm, indicating a large number o f conformational substates in solution. Upon polymerization, the mean distance decreased to 2.22 nm, but the width of the distribution did n ot change. Further addition of tropomyosin and troponin in the presenc e and absence of Ca2+ did not significantly change the mean distance b ut decreased the width of the distribution appreciably. Upon addition of S1, the mean distance increased by 0.38 nm, and the width of the di stribution decreased substantially.