Domain motion in actin observed by fluorescence resonance energy transfer.
Actin is composed of two well-separated globular domains which are further subdivided into two subdomains [Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C. (1990) Nature 347, 37-44]. Subdomains 1 and 2 constitute the small domain, and subdomains 3 and 4 comprise 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 measured by steady-state and time-resolved fluorometers. The adenine base is bound in a hydrophobic pocket between subdomains 3 and 4, and Tyr-69 is located at subdomain 2. In the present study, the adenine moiety was labeled with a fluorescence donor, epsilon ATP, and tyrosine-69 was labeled with the energy acceptor, dansyl chloride. Assuming the random orientation factor k2 = 2/3, the distance between epsilon-adenine moiety and dansyl chloride attached to Tyr-69 in G-actin was determined to be 2.46 nm from steady-state fluorescence measurements. The addition 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 other hand, time-resolved fluorescence energy transfer measurements have been used to investigate a distribution of distances for a donor-acceptor pair. In G-actin, the mean distance between probes was 2.79 nm with a full width at half-maximum of 3.91 nm, indicating a large number of conformational substates in solution.(ABSTRACT TRUNCATED AT 250 WORDS)
Miki
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Department of Applied Chemistry and BioTechnology, Fukui University, Japan.
Kouyama
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D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003619:Dansyl Compounds / Q000378:metabolism; D004735:Energy Transfer; D004985:Ethenoadenosine Triphosphate / Q000378:metabolism; D005453:Fluorescence; D005454:Fluorescence Polarization; D046911:Macromolecular Substances; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014335:Tropomyosin / Q000494:pharmacology; D014336:Troponin / Q000494:pharmacology
D000199:Actins; D003619:Dansyl Compounds; D004985:Ethenoadenosine Triphosphate; D046911:Macromolecular Substances; D014335:Tropomyosin; D014336:Troponin; C015195:1,N(6)-ethenoadenosine diphosphate; D000244:Adenosine Diphosphate; C029038:dansyl chloride; D002118:Calcium