Detection of conformational changes in actin by fluorescence resonance energy transfer between tyrosine-69 and cysteine-374.
The distance between 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride or DNS-Cl) attached to Tyr-69 and N-[[4-[4-(dimethylamino)phenyl]azo]phenyl]maleimide (DABMI) or N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide (DDPM) attached to Cys-374 in an actin monomer was measured to be 2.51 nm or 2.27 +/- 0.04 nm, respectively, by fluorescence resonance energy transfer. This distance does not change significantly when the actin monomer binds DNase I, when the monomer is polymerized, when the polymer interacts with myosin subfragment 1, or when it interacts with tropomyosin-troponin in the presence and absence of Ca2+. Changes in the distance were within 0.1 nm. The results indicate that the structure of the region involving Tyr-69 and Cys-374 is substantially rigid. A large blue shift (about 15 nm) of the fluorescence spectrum and a large increase (about 80%) in the fluorescence intensity of DNS-actin were observed when DNS-actin was denatured upon addition of EDTA. On the other hand, a red shift (about 7 nm) of the fluorescence spectrum and a large decrease (about 50%) in the fluorescence intensity were observed when DNS-actin was completely unfolded in 8 M urea. The results indicate that dansyl chromophore becomes less exposed to the aqueous environment by EDTA denaturation in contradiction to the case of intrinsic tryptophan residues in G-actin. Resonance energy transfer measurements showed that the distance between probes attached to Tyr-69 and Cys-374 on an actin monomer changes by 0.37 nm during EDTA denaturation, but that the distance becomes longer than 4.0 nm in 8 M urea in which no energy transfer is observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Miki
M
M
Department of Anatomy, University of Sydney, New South Wales, Australia.
D000199:Actins / Q000737:chemistry* / Q000302:isolation & purification; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003545:Cysteine; D003619:Dansyl Compounds; D004492:Edetic Acid / Q000494:pharmacology; D004735:Energy Transfer; D005456:Fluorescent Dyes; D008958:Models, Molecular; D009132:Muscles / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014443:Tyrosine; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D000199:Actins; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D014443:Tyrosine; C049748:4-dimethylaminophenylazophenyl-4'-maleimide; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D004124:p-Dimethylaminoazobenzene; D003545:Cysteine; C029038:dansyl chloride