Fluorescence energy transfer in myosin subfragment-1

Marsh, David J.; Lowey, Susan

doi:10.1021/bi00545a025

Cited by 94 publications

(50 citation statements)

References 58 publications

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“…The quantum yield of 175-IAEDANS bound to S1 in 30 mM KCl, 1.5 mM MgC12 and 20 mM Tris/HCl, pH 7.6 (buffer A) at 20°C was determined to be 0.44 in accordance with the value of 0.48 reported previously [32]. The quantum yield of FITC bound to actin in buffer A at 20 "C was determined to be 0.45 using FITC in 0.1 M NaOH as the standard which has a absolute quantum yield of 0.85 P31.…”

Section: Fluorescence Resonance Energy Transfersupporting

confidence: 87%

The recovery of the polymerizability of Lys-61-labelled actin by the addition of phalloidin. Fluorescence polarization and resonance-energy-transfer measurements

Miki

1987

Eur J Biochem

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Modification of Lys-61 in actin with fluorescein-5-isothiocyanate (FITC) blocks actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, [57][58][59][60][61][62]. FITC-labelled actin recovered its ability to polymerize on addition of phalloidin. The polymers had the same characteristic helical thread-like structure as normal F-actin and the addition of myosin subfragment-1 to the polymers formed the characteristic arrowhead structure in electron microscopy. The polymers activated the ATPase activity of myosin subfragment-I as efficiently as normal F-actin. These results indicate that Lys-61 is not directly involved in an actin-actin binding region nor in myosin binding site.From static fluorescence polarization measurements, the rotational relaxation time of FITC-labelled actin filaments was calculated to be 20 ns as the value reduced in water at 20°C, while any rotational relaxation time of 1,5-IAEDANS bound to Cys-374 on F-actin in the presence of a twofold molar excess of phalloidin could not be detected by static polarization measurements under the same conditions. This indicates that the Lys-61 side chain is extremely mobile even in the filamentous structure.Fluorescence resonance energy transfer between the donor 1,5-IAEDANS bound to SH1 of myosin subfragment-I and the acceptor fluorescein-5-isothiocyanate bound to Lys-61 of actin in the rigor complex was measured. The transfer efficiency was 0.39 f 0.05 which corresponds to the distance of 5.2 f 0.1 nm, assuming that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime and that the transfer occurs between a single donor and an acceptor.Bovine pancreatic DNase I depolymerizes F-actin and forms a 1 : l complex [I] with actin monomers. The threedimensional structure of the complex of actin and DNase I has been obtained by X-ray crystallography at 0.45-nm resolution [2]. According to this model, an actin subunit consists of a small and a large domain with the dimensions of 6.7 x 4.0 x 3.7 nm, the small domain containing the N-terminus. The phosphate of a bound nucleotide is located between the two domains. DNase I binds mainly to a loop region in the small domain of actin. On the other hand, chemical cross-linking methods revealed that residues at positions 50, 53, 61, 68 and/or 69 are within 0.3 nm of DNase I binding site [3]. These residues may be located near actin-actin binding site, since the chemical modifications of , Lys-61 [5] and impairs the polymerizability of actin without a gross conformational change such as detected by circular dichroic measurements.Phalloidin binds tightly to F-actin but not to G-actin [7, 81 and stabilizes the filamentous structure of actin not only towards depolymerizing conditions or agents but also towards heat denaturation and proteolytic degradation [9 -1 I]. The binding of phalloidin influences the polymerization of actin by stabilizing nuclei and polymers as they are formed [8] and also induces a conformational change in actin [12].The interaction of actin and myosin i...

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Section: Fluorescence Resonance Energy Transfersupporting

confidence: 87%

The recovery of the polymerizability of Lys-61-labelled actin by the addition of phalloidin. Fluorescence polarization and resonance-energy-transfer measurements

Miki

1987

Eur J Biochem

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“…A major difference between the two LCs lies in the ELC's proximity to myosin's motor domain, where the amino terminus ofthe myosin heavy chain abuts the ELC (3). This close interaction is reflected by the fact that MgATP hydrolysis in the motor domain caused a fluorophore bound to the ELC to decrease its signal with the same kinetics as the hydrolysis step (21,22). It is therefore not too surprising that ELC removal in turn affects the mechanical and kinetic properties of the myosin head.…”

Section: Discussionmentioning

confidence: 99%

The essential light chain is required for full force production by skeletal muscle myosin.

VanBuren

Waller

Harris

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

127

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Myosin, a molecular motor that is responsible for muscle contraction, is composed of two heavy chains each with two light chains. The crystal structure of subfragment 1 indicates that both the regulatory light chains (RLCs) and the essential light chains (ELCs) stabilize an extended a-helical segment of the heavy chain. It has recently been shown in a motility assay that removal of either light chain markedly reduces actin frament sliding velocity without a significant loss in actin-activated ATPase activity. Here we demonstrate by single actin frament force measurements that RLC removal has little effect on isometric force, whereas ELC removal reduces isometric force by over 50%. These data are interpreted with a simple mechanical model where subfragment 1 behaves as a torque motor whose lever arm length is sensitive to light-chain removal. Although the effect of removing RLCs fits within the confines of this model, altered crossbridge kinetics, as reflected in a reduced unloaded duty cycle, probably contributes to the reduced velocity and force production of ELC-deficient myosins.

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“…For example, we now know that Cys 126 is located at the interface of the RLC and the ELC; by engineering a Cys into an appropriate position on the ELC, it may be possible to detect a relative movement between the light chains upon ATP hydrolysis. It was previously shown that a probe bound to the endogenous COOH-terminal Cys of the ELC changed its fluorescence signal in parallel to the rate of myosin ATP hydrolysis (Marsh and Lowey, 1980;Marsh et al, 1982). This result is highly suggestive of a relative movement of the light chains within the neck region of myosin.…”

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confidence: 83%

Proximity Relationships between Engineered Cysteine Residues in Chicken Skeletal Myosin Regulatory Light Chain:

Wolff-Long

Tao

Lowey

1995

Journal of Biological Chemistry

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Resonance energy transfer was used to measure the distances between pairs of cysteines, Cys 2 and Cys 155and Cys 73 and Cys 155, in recombinant chicken skeletal myosin regulatory light chains in the free and bound states. The fluorescent and nonfluorescent probes N-iodoacetyl-N-(5-sulfo-1-naphthyl)ethylenediamine and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide were used as the donor and the acceptor, respectively. The distance between Cys 2 and Cys 155 was measured to be 35 and 30 Å in the absence and presence of myosin heavy chain, respectively, suggesting a slightly more compact structure for the light chain in the bound state. The distance between Cys 73 and Cys 155 measured in a similar manner was 31 and 30 Å in the free and bound states, respectively; this latter value is in good agreement with that derived from crystallographic structures. For heavy chain-bound light chains, no measurable distance changes were detected with the binding of ATP or actin. These results show that no gross structural changes occur within the regulatory light chain during the contraction cycle, but that resonance energy transfer between other sites could be used to monitor potential changes in the myosin head upon the binding of nucleotides and actin.Force generation in muscle results from a cyclic interaction of the myosin head (S1) 1 with actin. It is currently believed that S1 may change its orientation relative to the actin filament during this process and thereby produce a displacement of ϳ50 -100 Å, the so-called "working stroke" (Huxley, 1969;Huxley and Simmons, 1971). A persistent problem with the "rotating cross-bridge model" has been the failure to detect any large conformational change in the head by most biophysical methods. It has been suggested that the reactive Cys 707 on the heavy chain, the site of labeling for most spectroscopic probes, may not be particularly sensitive to angular changes, and the portion of S1 adjacent to the rod might undergo more pronounced structural changes during hydrolysis (Huxley and Kress, 1985). We have therefore focused our efforts over the last few years on preparing Cys mutants of the regulatory light chain (RLC), a subunit which is readily exchanged into myosin, and can therefore provide a means to introduce probes into the COOH-terminal region of the myosin head (Saraswat and Lowey, 1991;Saraswat et al., 1992). Moreover, this approach avoids modification of the reactive heavy chain Cys residues, SH1 and SH2, which can have adverse effects on the enzymatic activity and motor properties of myosin (Root and Reisler, 1992).Several earlier studies have used labeled light chains to detect structural changes in the myosin head. By incorporating a 1,5-IAEDANS-labeled RLC into smooth muscle myosin, Morita et al. (1991) were able to show by fluorescence anisotropy that a change in conformation at the head/rod junction can be correlated with the transition from a folded to an extended myosin molecule. Hambly et al. (1991) exchanged a spin-labeled light chain into skinned skeletal muscle ...

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Fluorescence energy transfer in myosin subfragment-1

Cited by 94 publications

References 58 publications

The recovery of the polymerizability of Lys-61-labelled actin by the addition of phalloidin. Fluorescence polarization and resonance-energy-transfer measurements

The recovery of the polymerizability of Lys-61-labelled actin by the addition of phalloidin. Fluorescence polarization and resonance-energy-transfer measurements

The essential light chain is required for full force production by skeletal muscle myosin.

Proximity Relationships between Engineered Cysteine Residues in Chicken Skeletal Myosin Regulatory Light Chain:

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