Spatial relationship between a fast-reacting thiol and a reactive lysine residue of myosin subfragment 1.
Fluorescence energy transfer was used to examine the spatial proximity between two key side chains in myosin subfragment 1 (S-1), viz., the reactive thiol (SH1) located on the C-terminal 20K tryptic fragment and the reactive lysyl (RLR) on the N-terminal 27K tryptic fragment of S-1 heavy chain. S-1 was specifically labeled at SH1 with an energy donor, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS), and at RLR with an energy acceptor, 2,4,6,-trinitrobenzenesulfonate (TNBS). Prior blocking of SH1 with AEDANS increased the pK of RLR from 9.04 to 9.42. Trinitrophenylation of SH1-blocked S-1 was about 50% slower and sharply reduced the Ca2+ ATPase activity. Reciprocally, blocking of RLR with TNBS slowed the rate of reaction of SH1 and AEDANS by 40-60%. Addition of the second label does not grossly alter the conformation resulting from the first label. S-1 labeled at RLR with TNBS and at SH1 with optically inert iodoacetamide shows the same TNP difference spectrum +/- MgADP (lambda min 365 nm) as S-1 with S 1 free. Also, S-1 labeled at SH1 with AEDANS and at RLR with an optically inert methyl group shows the same AEDANS emission spectrum (lambda em max 475 nm), excited-state lifetime (tau = 20.3 ns) and rotational correlation time (phi = 106 ns) as S-1 with RLR free. When the decrease of either the quantum yield or the excited-state lifetime of the donor in the absence and presence of the acceptor was measured, the energy transfer efficiency was found to be 70%. The apparent interchromophore distance was calculated to be 2.6 nm through the use of the Förster equation with an uncertainty of less than 12%.
D000252:Calcium-Transporting ATPases; D004735:Energy Transfer; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014301:Trinitrobenzenes / Q000494:pharmacology; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology
D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D014301:Trinitrobenzenes; C006421:1,5-I-AEDANS; D014302:Trinitrobenzenesulfonic Acid; D009218:Myosins; D000252:Calcium-Transporting ATPases; D008239:Lysine