Spatial relationship between a fast-reacting thiol and a reactive lysine residue of myosin subfragment 1

• Published in: Biochemistry (Easton) 1982-10, Vol.21 (22), p.5661-5668
• Main Authors: Takashi, Reiji, Muhlrad, Andras, Botts, Jean
• Format: Article
• Language: English
• Subjects: Calcium-Transporting ATPases; conformational analysis; Energy Transfer; fluorescence; Kinetics; Lysine - metabolism; muscles; myosin; Myosins - metabolism; Naphthalenesulfonates - pharmacology; Peptide Fragments - metabolism; Protein Conformation; rabbits; Spectrometry, Fluorescence; Sulfhydryl Compounds - metabolism; Trinitrobenzenes - pharmacology; Trinitrobenzenesulfonic Acid - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00265a042

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%.