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

Abstract

Förster energy transfer was used to measure the distance between reporter groups on the two reactive thiols of 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 light chains. The [14C]-DDPM-modified alkali light chains were replaced with unmodified light chains by the exchange procedure of Wagner and Weeds [Wagner, P.D., & Weeds, A. G. (1977) J. Mol. Biol. 109, 455-473]. Subfragment 1 labeled with 1,5-IAEDANS and then with DDPM exhibited two 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 A 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 A. This observation is consistent with the proposal that nucleotide binding juxtaposes SH1 and SH2 to enhance their cross-linking with various bifunctional reagents [Burke, M., & Reisler, E. (1977) Biochemistry 16, 5559-5563; Wells, J. A., & Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970].