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].

Steady-state fluorescence polarization techniques were used to study the binding of initiation factor 3 (IF3) to 30S ribosomal subunits. Covalent fluorescent derivatives of IF3 were prepared by treating the pure protein with fluorescein isothiocyanate. The fluorescein-labeled IF3 (F-IF3) contained 0.8--1.7 dye molecules per protein. Polyacrylamide gel electrophoretic analysis of the derivatized forms is consistent with the view that the probe is randomly attached, presumably to lysine epsilon-amino groups. The activity of F-IF3 is not impaired in assays for binding to 30S ribosomal subunits or in promoting formylmethionyl-tRNA binding to 70S ribosomes. Fluorescence polarization values were measured at different F-IF3 and 30S ribosomal subunit concentrations, and the association constant and number of binding sites were calculated. In buffer containing 10 mM magnesium acetate and 100 mM ammonium chloride, the association constant is (3.1 +/- 1.4) x 10(7) M-1, and the number of ribosomal binding sites is 1.2 +/- 0.2. The value for the association constant varies inversely with the ammonium chloride and magnesium acetate concentrations by a small amount. Competition studies show that nonderivatized IF3 binds to 30S ribosomal subunits with the same affinity as F-IF3. Therefore, the association constants measured for F-IF3 are valid for IF3 as well.

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