Measurement of interprotein distances in the acto-subfragment 1 rigor complex.
Using enzymatic labeling, we have conjugated the fluorescence probe dansylcadaverine (DNC) to Gln-41 of rabbit skeletal muscle actin with the intention of utilizing the dansyl chromophore as a donor in fluorescence resonance energy transfer (FRET) distance measurements. The fluorescence decay of DNC-actin was found to consist of two decay constants (8.23 and 21.2 ns) that were associated with two different but partially overlapping spectra of the dye. Three different chemical points on myosin subfragment 1 (S1) were labeled with suitable acceptors: reactive thiol 1 (SH1) and Cys-136 on LC3 were modified with tetramethylrhodamine 5- (and 6-) iodoacetamide (ITMR); Lys-83 (RLR) was derivatized with trinitrobenzenesulfonate. In the rigor complex of the two labeled proteins, fluorescence resonance energy transfer took place, the efficiency of which was 10.9, 9.28, and 3.73% for the transfer from Gln-41 to SH1, Cys-136 (LC3), and RLR, respectively. The limits of the Förster critical distance for each pair were obtained from the analysis of the polarization spectra of the donor and of the acceptors. The kappa 2(2/3) distances from actin Gln-41 to the three points on S1 were 63, 66, and greater than 37 A for SH1, Cys-136 (LC3), and RLR, respectively.
Takashi
R
R
Cardiovascular Research Institute, University of California, San Francisco 94143.
Kasprzak
A A
AA
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives / Q000378:metabolism; D004396:Coloring Agents; D005456:Fluorescent Dyes; D007460:Iodoacetamide / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles / Q000378:metabolism*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D012235:Rhodamines / Q000378:metabolism
D000199:Actins; D004396:Coloring Agents; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; D012235:Rhodamines; C005358:tetramethylrhodamine; D009218:Myosins; C008542:monodansylcadaverine; D002103:Cadaverine; D007460:Iodoacetamide