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

Abstract

Fluorescence energy transfer was used to examine the spatial proximity between 2 key side chains in [rabbit] myosin subfragment 1 (S-1), i.e., the reactive thiol (SH1) located on the C-terminal 20K [kilodalton] tryptic fragment and the reactive lysyl (RLR) on the N-terminal 27K tryptic fragment of S-1 H 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 9.04-9.42. Trinitrophenylation of SH1-blocked S-1 was .apprx. 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 2nd label does not grossly alter the conformation resulting from the 1st label. S-1 labeled at RLR with TNBS and at SH1 with optically inert iodoacetamide sohws the same TNP difference spectrum .+-. MgADP (.lambda.min 365 nm) as S-1 with SH1 free. S-1 labeled at SH1 with AEDANS and at RLR with an optically inert methyl group shows the same AEDANS emission spectrum (.lambda.emmax475 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 70%. The apparent interchromophore distance was calculated to be 2.6 nm through the use of the Forster equation with an uncertainty of < 12%.