Fluorescence energy transfer between the myosin subfragment‐1 isoenzymes and F‐actin in the absence and presence of nucleotides

The unique fast‐reacting cysteine residue (SH1) of myosin subfragment 1 (S1), prepared by chymotryptic digestion, and cysteine 373 of actin have been labelled selectively with the fluorescent probes, N‐(bromoacetyl)‐N′‐(1‐sulpho‐5‐naphthyl)ethylenediamine (1, 5‐BrAEDANS) and 5‐(iodoacetamido)fluorescein (5‐IAF), whose spectral properties render them a particularly effective donor‐acceptor pair in fluorescence energy transfer studies. The transfer efficiency of 40–45% represented a spatial separation of the chromophores of about 5 nm, which is in reasonable agreement with the value of 6 nm reported earlier for similarly labelled S1, prepared by papain digestion, and actin [Takashi, R. (1979) Biochemistry, 18, 5164–5169]. This transfer efficiency did not change when the doubly‐labelled binary complex was formed: (1) with acto‐S1(A1) or acto‐S1(A2) at 10–200 mM KC1, pH 7–8 and different buffer conditions; (2) with either S1 isoenzyme and regulated actin (i. e. actin with tropomyosin and troponin) both in the presence and absence of Ca2+ or when the donor and acceptor attachment sites were reversed. Analysis of donor and acceptor polarized fluorescence showed that the chromophores are not randomly orientated (i.e. χ2≠ 2/3), but they do have some motion relative to either protein. From a knowledge of the limiting values for χ2, the intersite distance for donor and acceptor chromophores was calculated to be in the range 3.9–6.7 nm.Addition of MgATP to the doubly‐labelled acto‐S1 complex eliminated energy transfer but this was recovered when ATP hydrolysis was completed. By utilizing the known binding constants between S1, actin and either MgADP or MgAdoPP[NH]P (magnesium adenosine 5′‐[βγ‐imido]triphosphate) [Konrad, M. and Goody, K. (1982) Eur. J. Biochem. 128, 547–555; Greene, L. E. and Eisenberg, E. (1980) J. Biol. Chem. 255, 543–548], the concentrations of all species present at equilibrium were determined. Experimental conditions were chosen to maximise the amount of ternary acto‐S1‐nucleotide complex (∼ 50%) and minimise the amount of binary complex (≤ 2%). The spatial separation of the chromophore interaction sites in the ternary complex was found to be the same with both nucleotides and indistinguishable from that found with the binary complex. A similar strategy was employed to compare the conformations of the binary and ternary complexes by 1H‐NMR spectro‐scopy. In these experiments about 90% of the S1 was in the form of the ternary complex. There was no noticeable change in the acto‐S1 spectra upon addition of either MgAdo PP[NH]P or MgADP. These observations support the conclusion that there is no large change in structure in the ‘rigor’ binary acto‐S1 complex when it binds either ADP or Ado PP[NH]P.