Reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with myosin subfragment one: evidence for formation of a single protein disulfide with trapping of metal nucleotide at the active site

Wells, James A.; Yount, Ralph G.

doi:10.1021/bi00549a030

Cited by 92 publications

(59 citation statements)

References 25 publications

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“…1B). No alteration of the elevated Ca2+-ATPase of AEDANS-S-1 and N2ph-S-1 occurred in the absence of Mg2+-nucleotide, whereas a progressive increase of the original Ca2+-ATPase of the native S-1 control was observed, as expected, owing to the rapid primary reaction of Nbs2 with the free SH1 (9). In contrast, the addition of Mg2+-nucleotide resulted in a rapid inactivation of the premodified S-1 derivatives, but at a rate lower (t/2 = 10-15 min) than with Ph(NMal)2; during the first 70 min of reaction, Ph(NMal)2 seems effective only in the presence of Mg2 -nucleotide.…”

Section: Resultssupporting

confidence: 73%

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

Chaussepied¹,

Mornet²,

Kassab³

1986

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

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When myosin subfragment 1 derivatives in which the reactive sulfhydryl SH1 has been blocked react with N,N'-p-phenylenedimaleimide or 5,5'-dithiobis(2-nitrobenzoic acid), the reactive sulfhydryl group SH2 of the 20-kDa domain is crosslinked with a thiol of the 50-kDa domain of the heavy chain. The crosslink induces the stable trapping of a significant amount of Rabbit skeletal muscle actin was prepared as described by Eisenberg and Kielley (17), and its concentration was estimated by using A19i, = 11.0.Crosslinking Reactions. The reaction of 1.3-fold excess Ph(NMal)2 in 50 mM Hepes (pH 8.0) was done with 50 ,uM modified or native enzyme in the presence or absence of 2.5 mM Mg2+-ATP or Mg2+-ADP at 4°C. The crosslinking of S-1 with Nbs2 was performed under similar conditions, using a 5-fold excess of reagent. The reactions were quenched and the protein derivatives were purified by filtration over Sephadex G-50 (20 x 1.5 cm) in 50 mM Hepes, pH 7.0/200 mM KCl/0.5 mM NaN3 at 40C. NaDodSO4/PAGE. Tryptic fragments of modified S-1 were separated by electrophoresis in 0.1% NaDodSO4/polyacrylamide slab gels containing a 5-18% (wt/vol) gradient of acrylamide (18). The running buffer was 50 mM Tris/100 mM boric acid (pH 8.0) (19). The densitometric scanning of the gels was carried out with a computerized model CS930 Shimadzu (Kyoto, Japan) gel scanner. The following were used as molecular mass markers: S-1 heavy chain (95 kDa), S-1 light chain 1 (LC1, 25 kDa), S-1 light chain 3 (LC3, 17 kDa), the three fragments obtained by tryptic cleavage of S-1 (50 kDa, 27 kDa, and 20 kDa), and actin (42 kDa).Specific Modifications of S-1 Thiols. The specific fluorescence labeling of SH1 with 5-[2-(iodoacetyl)aminoethyl]aminonaphthalene-1-sulfonic acid (1,5-IAEDANS) was done according to the procedure of Takashi et al. (20). Under our conditions, 1.1 mol of the dye was incorporated per mol of S-1. Fluorescence gel scanning allowed us to quantify the labeling ratio as 0.2 in the light chains and 0.9 in the heavy chain.The specific chemical modifications of SH1 by 2,4-dinitrofluorobenzene (N2ph-F) and iodoacetamide were carried out as described by Reisler et al.

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Section: Resultssupporting

confidence: 73%

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

Chaussepied¹,

Mornet²,

Kassab³

1986

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

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“…Such surmises were much enhanced by Reisler et al (63), who discovered the first topographical crosslink-Cys-697 to Cys-707-and showed that it can be fixed at various lengths. More recently, Wells and Yount (64) showed that when this crosslink is made in the presence of bound ADP it retards the escape of the then trapped ADP but that subsequent actin binding releases the trap. That bound nucleotide enters a "pocket" is also shown by the observations of Ando et al (65).…”

Section: Structural Information Of Other Kindsmentioning

confidence: 99%

On the origin and transmission of force in actomyosin subfragment 1.

Botts

Thomason

Morales

1989

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

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A proximity map showing the three-dimensional arrangement of 12 chemically defined points in actomyosin subfragment 1 is developed and roughly correlated with published electron microscope reconstructions of others. Several additional points and topological relationships in the primary polypeptide chain folding are assimilated into this model. Certain crossliikings and distance change observations are interpreted as indicators of transmission of force/displacement between the nucleotide-binding and an actin-binding site-i.e., as indications of how energy is transduced in this system.It is reasonable to think that contractile force arises because the equilibrium geometrical relations between actin and the subfragment 1 (S-1) segment of myosin are different for each enzymatic intermediate occupying an ATPase site of S-1 that is remote from the (S-1)-actin interface(s). Then the operation of the enzymatic cycle compels operation of a cycle of relations between S-1 and actin. If changes in these relations are resisted by (surmountable) external forces, the cycle of relations is a "work cycle" performing external work. In a repetitive system, chemical free energy is thus continuously transduced into mechanical work. One has to say how the binding of an enzymatic intermediate determines an actinbinding relation 5 nm away. We have suggested that binding of the intermediate at the ATPase distorts the local S-1 structure and that this distortion is mechanically transmitted to the distant actin-binding site. These ideas have been developed in previous papers (1-3). This paper fleshes out such ideas with recent observations. Structural Information froin Fluorescence Resonance Energy Transfer (FRET) Mapping and Electron Microscopy (EM)We begin by exploring the structure that may transmit effects between the nucleotide-binding site (N site) and the actinbinding site(s) (A site).A Since crystallography is still unavailable, we resort to melding lesser methods. As shown previously, chemically defined points in acto-(S-1) can be pairwise labeled by fluorophores. From FRET between such a pair one can estimate the interpoint distance, and by repetition of the procedure one can build up a lattice of acto-(S-1) points (2, 5). The improvement offered by the Dale et al. (6) analysis was previously incorporated (2, 7), but since 1984, other developments have appeared (see below). Though we cite improvements we must also emphasize the limitations of proximity mapping inherent in our results. For example, every point lattice has at least one unresolvable "mirror image" ambiguity. More serious is a shortage of necessary distance measurements. Although n(n -1)/2 distances are displayed in a complete lattice of n points, only 4n -10 distances are required to construct the lattice (2). But in our case even this smaller number has not yet been obtained. The missing distances have been assigned by using extraneous information of variable reliability. For example, chemical information may favor the proximity of two points, sequence loc...

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“…In the experiments reported here variation of the protein concentration by more than 100-fold (0.4 -60 1 M ) did not affect the measured affinities. The affinity of Mg-AdoPP[NH]P to myosin subfragment-I lies in the range of published values obtained by direct methods of 0.8-2.1 x lo6 M-' [I, [24][25][26], as well as by indirect methods, such as fluorescence, which gave 3.3 x lo6 M -' [27] and higher than 2 x lo6 M-' [28], calorimetry with 1.7 x 105 M-' [29] or in competition experiments with 1, For Mg-pyrophosphate binding to subfragment-I using direct are reported, which are also close to our value. For both the ligands Mg-AdoPP[NH]P and Mg-pyrophosphate no temperature dependence was observed for the affinity constants between 2 "C and 25°C [1,25].…”

Section: Discussionmentioning

confidence: 75%

The Role of Magnesium in Binding of the Nucleotide Polyphosphate Chain to the Active Site of Myosin Subfragment‐1

Schaub

Watterson

Loth

et al. 1983

European Journal of Biochemistry

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The binding of adenosine 5'-[P,y-imidoltriphosphate, pyrophosphate and triphosphate to the active site of myosin subfragment-I was assessed in the presence and absence of Mg2+ by direct and indirect methods. In addition, the affinity and stoichiometry of Mg2+ in the ternary complexes formed by protein, Mg2+ and each of these phosphate compounds have been determined. As direct methods, equilibrium dialysis, sedimentation and quantitative affinity chromatography were used in conjunction with the indirect method of monitoring reactivity changes of the critical thiol-1 and thiol-2 groups, which occur upon binding of the ligands at the active site. There was good agreement between the results yielded by the different methods. All three phosphate compounds alone bind just one molecule per isolated myosin head portion with similar affinities lying in the range 1-4 x lo3 M-'. Again only one molecule/head portion binds when they exist in the form complexed with Mg2+, but now show much higher affinities of between 1 O6 -lo7 M-'. In all cases Mg2+ was found to be associated in the ternary complexes with the very high affinity of lo8 -lo9 M-'. It is postulated that this ion plays a prominent role in fixing the phosphate chain in the myosin active site. In contrast, Mg2+ scarcely affects the affinity of ADP and shows only a low affinity around 4 x lo4 M-' in the ternary complex Isolated myosin subfragments-I, obtained by proteolytic digestion of the head portions from the duplex myosin molecule, are assumed to possess an identically functioning active site each. However, in attempts to label the site covalently with the use of the affinity probes 6,6'-dithiobis (inosine 5'-[p,yimidoltriphosphate) [I] and 2',3'-isopropylidene-adenosine 5'-triphosphate [2], the markers were found at sites in addition to the hydrolytic one. Furthermore, reversible binding studies with ATP in the absence of divalent cations have led to the interpretation of a nonhydrolytic second site for ATP cxisting on myosin subfragment-I [3], which was postulated to have a regulatory function. We have recently shown that there is only one homogeneous binding site for the product ADP in both the presence and absence of divalent cations on subfragment-1 by employing direct and indirect methods [4]. It was also demonstrated that Mg2+ has only a relatively low affinity to the binary protein-ADP complex. We have now extended these studies to include the non-hydrolyzable substrate analogue adenosine 5'-[P,y-imidoltriphosphate (AdoPP[NH]P) [ 5 ] , as well as pyrophosphate and triphosphate. In addition, the role of Mg2' in the binding of these phosphate chains to the active site was investigated.In the absence of divalent cations these ligands have very low affinities, so that reliable methods must be employed for these conditions. The more conventional direct methods, equilibrium dialysis and sedimentation, are no longer accurate enough for measuring affinities below lo4 M -' [4]. Therefore we included studies using quantitative affinity chromatography [6] and the...

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Reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with myosin subfragment one: evidence for formation of a single protein disulfide with trapping of metal nucleotide at the active site

Cited by 92 publications

References 25 publications

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

On the origin and transmission of force in actomyosin subfragment 1.

The Role of Magnesium in Binding of the Nucleotide Polyphosphate Chain to the Active Site of Myosin Subfragment‐1

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