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

Abstract

The unique fast-reacting cysteine residue (SH,) of myosin subfragment 1 (Sl), prepared by chymotryptic digestion, and cysteine 373 of actin have been labelled selectively with the fluorescent probes, N-(bromoacety1)-N'-(1-sulpho-5-naphthy1)ethylenediamine (1, 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][41][42][43][44][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, 5164-51691. This transfer efficiency did not change when the doubly-labelled binary complex was formed: (1) with acto-Sl(A1) or acto-Sl(A2) at 10-200 mM KCl, 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. x2 # 2/3), but they do have some motion relative to either protein.From a knowledge of the limiting values for x2, 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 Biol. Chem. 255,, the concentrations of all species present at equilibrium were determined. Experimental conditions were chosen to maximise the amount of ternary acto-S1-nucleotide complex (z 50%) and minimise the amount of binary complex ( 5 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 'H-NMR spectroscopy. 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 MgAdoPP[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 AdoPP[NH]P.The interaction between actin and myosin is central to the energy transduction process in muscle. However, whereas actin is a highly conserved protein [I], the subunit of the hexameric myosin molecule can differ widely both between tissues and within a single tissue [2]. It is thought that this variation in the sequence and composition of the myosin subunits is largely responsible for the different contractile properties exhibited by different muscle (and non-muscle) tiss...

Decavanadate Binding to a High Affinity Site near the Myosin Catalytic Centre Inhibits F-Actin-Stimulated Myosin ATPase Activity

“…The formation of the complex between actin and S1 can also be monitored by fluorescence energy transfer (48,51,52). To this end, we have labeled S1 with IAF (as the fluorescent donor) and F-actin with eosin iodoacetamide (eosin-F-actin).…”

Localization of two myosin‐subfragment‐1 binding contacts in the 96–132 region of actin subdomain‐1

“…S-1(A,) and S-1(A,) isoenzymes were isolated by chromatography on a SP-Trisacryl M column (Trayer and 'Trayer, 1983). The trypsin derivative (27 -50-220-kDa) S-1 was prepared as described by Mornet et al (1979).…”

The widespread distribution of α‐N‐trimethylalanine as the N‐terminal amino acid of light chains from vertebrate striated muscle myosins

“…S 1 was prepared from rabbit fast-twitch muscle myosin by chymotryptic digestion [ 191 as modified in [20]. The pure isoenzymes Sl(A1) and SI(A2), were isolated by a single pass down SE-cellulose as described in [21]. Cardiac myosin was prepared from bovine ventricles by the procedure outline in [18], except that the minced muscle washed with 0.04 M KCI before extraction and after each subsequent precipitation step in order to remove the red colouration.…”

“…mol-' of light chains, two associated with each of the head regions of the molecule [I, 21. These light chains are of two types: the so-called LC2-light chains, which are phosphorylatable in vertebrate myosins [3], can be dissociated from myosin by 5,5'-dithiobis(2-nitrobenzoic acid) [I] and are analogous to the Ca-binding regulatory light chains in invertebrate myosins [4], and the socalled alkali light chains, A 1(LC l ) and A2(LC3) in vertebrate fast-twitch muscle, which are analogous to the SH-light chain of scallop myosin [4].…”