Foerster energy transfer measurements of thiol 1 to thiol 2 distances in myosin subfragment 1

Abstract

Foester energy transfer was used to measure the distance between reporter groups on the 2 reactive thiols of [rabbit muscle] myosin, SH1 and SH2 and to detect changes in this distance upon binding of nucleotide. SH1 was labeled with the fluorophore 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (1,5-IAEDANS) and SH2 with the chromophoric acceptor N-[4-(dimethylamino)-3,5-dinitrophenyl]-maleimide (DDPM). Peptide studies verified that [3H]-1,5-IAEDANS reacted specifically with SH1, while [14C]DDPM labeled both SH2 and the alkali L chains. The [14C]-DDPM-modified alkali L chains were replaced with unmodified L chains by the exchange procedure of Wagner and Weeds. Subfragment 1 labeled with 1,5-IAEDANS and then with DDPM exhibited 2 fluorescence lifetimes, 20.6 (AEDANS-SF1, unquenched) and 9.3 ns (AEDANS-SF1, quenched by DDPM). The latter lifetime decreased to an average of 2.85 ns after the addition of MgAMP-PNP, MgADP or MgPPi (no change with MgAMP), indicating that the distance between the donor and acceptor decreased. An R0 of 29 .ANG. was calculated for the AEDANS/DDPM system assuming random orientation of the donor/acceptor pair. The decrease in the observed lifetimes upon the addition of Mg nucleotide corresponds to a change in the donor-acceptor distance from 28 to 21-22 .ANG.. This observation is consistent with the proposal that nucleotide binding juxtaposes SH1 and SH2 to enhance their cross-linking with various bifunctional reagents.