Identification of a surface actin-binding site on myosin

Prince, Hrh; Levine, Barry A.; Trayer, Ian P.

doi:10.1016/0167-4838(82)90399-5

Cited by 21 publications

(13 citation statements)

References 16 publications

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“…Furthermore, the presence of a particular alkali light chain associated with the myosin head appears to have a definite influence on the affinity of the heads for actin [19-211, possibly suggesting a role for the N-terminal 41 amino acid residues of the A1 light chain. Recent 'H-NMR studies from our laboratory [22] support this proposition. The 41 -residue tail of the A1 light chain is clearly visible in the SI(A1) spectrum, demonstrating its high segmental mobility, which is considerably constrained upon interaction with actin.…”

supporting

confidence: 60%

“…Our experiments have assumed that there is enough information in the myosin head region alone to facilitate any conformational changes in S1 which occur in the acto-S1 complex on binding nucleotide. Although it has been proposed that the myosin head maintains a constant orientation during the power stroke of muscle contraction and some other part of myosin shortens [69], the demonstration of work production by actomyosin machines using only the soluble S1 fragment seem to restrict conformational changes necessary for force generation to this area of the molecule [70J Furthermore, the finding that virtually all the internal mobility of the myosin molecule is confined to the S1 region and that this is severely constrained by interaction with actin supports this argument [21,22,71].…”

Section: Fluorescence Energy Transfer In the Ternary Acto-s1adp Complexmentioning

confidence: 54%

“…'H-NMR is a non-invasive technique which is well recognized as giving information about the structure of proteins in solution and has been used successfully to investigate the structural changes in the head region of myosin on binding to actin in the binary complex [21,22,711. The higher protein concentrations used in NMR studies also enable a larger proportion of S1 to be driven into the acto-S1-nucleotide complex ( 2 90 %).…”

Section: Fluorescence Energy Transfer In the Ternary Acto-s1adp Complexmentioning

confidence: 99%

“…Shifts were measured relative to internal sodium 3-trimethylsilyl-(2,2,3,3-*H)propionate. The proteins were prepared as indicated in Materials and Methods and dialysed extensively against the above buffers in deuterium oxide at the concentrations indicated; molecular masses were taken as 110 kDa and 42 kDa for Sl(A1) and actin respectively, Spectra were recorded in a 300-MHz Bruker spectrometer operated in the Fourier-transform mode as indicated in [22] ATP analogues and their ability to induce conformational changes in the actinomyosin complex and how this might relate to bound cross-bridge states is reviewed in [91].…”

Section: Fluorescence Energy Transfer In the Ternary Acto-s1adp Complexmentioning

confidence: 99%

“…The 41 -residue tail of the A1 light chain is clearly visible in the SI(A1) spectrum, demonstrating its high segmental mobility, which is considerably constrained upon interaction with actin. Furthermore, all vertebrate striated muscle light chains of the A1 type appear to contain a novel N-terminal amino acid, N-trimethylalanine, which is involved in direct interaction with actin [22,23]. In other words, the structural differences between the two S1 isoenzymes appear to be expressed in their binary complexes with actin.…”

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confidence: 99%

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

Trayer

1983

European Journal of Biochemistry

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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...

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supporting

confidence: 60%

Section: Fluorescence Energy Transfer In the Ternary Acto-s1adp Complexmentioning

confidence: 54%

Section: Fluorescence Energy Transfer In the Ternary Acto-s1adp Complexmentioning

confidence: 99%

Section: Fluorescence Energy Transfer In the Ternary Acto-s1adp Complexmentioning

confidence: 99%

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confidence: 99%

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