Defining the “fast-reacting” thiols of myosin by reaction with 1,5 IAEDANS

Takashi, Reiji; Duke, Joseph A.; Ue, Kathleen; Morales, Manuel F.

doi:10.1016/0003-9861(76)90509-9

Cited by 74 publications

(64 citation statements)

References 13 publications

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“…In a first step, the reactive SHi group of chymotryptic S-1 (containing LC1 and LC3) was protected by three reagents that react quantitatively and specifically with this thiol. These were the fluorescent dye 1,5-IAEDANS (20), iodoacetamide (22), and N2ph-F (21); N2ph-F allows a reversible blocking of the SH1 thiol (25). The isolated S-1 derivatives exhibited about 5% the K+-ATPase activity of unmodified S-1, whereas their Ca2+-ATPase was enhanced to 350-450% the control value.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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: Resultsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

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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“…S1 modifications were carried out with an Cys707-specific fluorescent reagent, I-AE-DANS (Takashi et al, 1976) with a 1:10 ratio of reagent/Sl. (Table 1).…”

Section: Modification Ofmentioning

confidence: 99%

Effect of Complexes of ADP and Phosphate Analogs on the Conformation of the Cys707‐Cys697 Region of Myosin Subfragment 1

Phan

Peyser

Reisler

et al. 1997

European Journal of Biochemistry

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Recent crystallographic studies have suggested structural differences between the complexes of S1 ˙ Mg ˙ ADP with the phosphate analogs aluminium fluoride (AlF−4), vanadate (VO3‐4) and beryllium fluoride (BeFx) [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960–8972; Smith, R. & Rayment, I. (1996) Biochemistry 35, 5404–54171. In this work, chemical modifications, namely labeling of Cys707 (the reactive SH1 thiol) and Cys707 – Cys697 (SH1‐SH2) cross‐linking, were used to compare the S1 ˙ ADP ˙ BeFx, S1 ˙ ADP ˙ AlF−4 and S1 ˙ ADP ˙ VO3‐4 complexes with specific states of the myosin‐ATPase pathway. Modification of Cys707 with the fluorescent monofunctional reagents 7‐diethylamino‐3‐(4′‐maleimidylphenyl)‐4‐methylcoumarin and N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphtyl)ethylenediamine has shown that the reactivity of the SH1 group depends on the nucleotide bound to S1. The observed rates of Cys707 modification at 20°C lead to the conclusion that S1˙ ADP ˙ BeFx is similar to S1*˙ ATP, while S1 ˙ ADP ˙ AIF−4 and S1 ˙ ADP ˙ VO43‐ are more similar to S1**˙ ADP ˙ Pi. The conformations of the analog states were also compared by monitoring the dissociation of the fluorescent nucleotide analog 1‐N6‐ethenoadenosine diphosphate (ADP[C2H2]) from the active site of Cys707‐modified (by N‐ethylmaleimide) and Cys707‐Cys697‐cross‐linked (by N,N′‐p‐phenylene dimaleimide) S1 ˙ ADP[C2H2] ˙ AlF−4 and S1 ˙ ADP[C2H2] ˙ BeFx. Our results suggest that the conformations of the S1 ˙ ADP ˙ AlF−4, S1 ˙ ADP ˙ VO3‐4 and S1 ˙ ADP ˙ BeFx, complexes in the Cys707–Cys697 region are distinct from each other, with the former two at least partially resembling the S1**˙ ADP ˙ Pi state, while the latter is similar to the prehydrolyzed S1*˙ ATP state.

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“…Properties of BD-Alkylation of S-1 with IAA and IAEDANS invariably results in the modification of only SH1 (20,26,27). Although chemical properties of BD had scarcely been studied, the reagent was expected to be useful as a specific fluorescent label for SH1 of S-1.…”

Section: Resultsmentioning

confidence: 99%

“…Several environmentally sensitive fluorescent probes are known to label conveniently only SH1 (14,20,26). In contrast to this, thus far little has been known about such probes for SH2 (20).…”

Section: Discussionmentioning

confidence: 99%

ATP-induced Opposite Changes in the Local Environments around Cys697 (SH2) and Cys707 (SH1) of the Myosin Motor Domain Revealed by the Prodan Fluorescence

Hiratsuka

1999

Journal of Biological Chemistry

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To obtain a consistent view of the nucleotide-induced conformational changes around Cys 697 (SH2) and Cys 707(SH1) in skeletal myosin subfragment-1 (S-1), the two thiols were labeled with the same environmentally sensitive fluorophore, 6-acyl-2-dimethylaminonaphthalene group, using 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AD) and 6-bromoacetyl-2-dimethylaminonaphthalene (BD), respectively. The resultant fluorescent derivatives, AD-S-1 and BD-S-1, have the same fluorophore at either SH2 or SH1, which was verified by inspections of changes in the ATPases and the localization of fluorescence after tryptic digestion and CNBr cleavage for the two derivatives. Especially, AD was found to be a very useful fluorescent reagent that readily reacts with only SH2 of S-1. Measurements of the nucleotideinduced changes in fluorescence emission spectra of AD-S-1 and BD-S-1 suggested that during ATP hydrolysis the environment around the fluorophore at SH2 is very distinct from that around the fluorophore at SH1, being defined as that the former has the hydrophobic and closed characteristics, whereas the latter has the hydrophilic and open ones. The KI quenching study of the fluorescence of the two S-1 derivatives confirmed these results. The most straightforward interpretation for the present results is that during ATP hydrolysis, the helix containing SH2 is buried in hydrophobic side chains and rather reinforced, whereas the adjacent helix containing SH1 moves away from its stabilizing tertiary structural environment.The structural element of the molecular motor myosin is the subfragment-1 (S-1) 1 moiety that contains the sites responsible for the ATP hydrolysis and binding of actin (1). A striking feature of the S-1 structure is a long helix spanning 85 Å that is stabilized by interactions with the myosin light chains (2). It has been proposed that this light chain-binding domain acts as a semi rigid "lever arm" to amplify and transmit conformational changes in the ATP and actin binding sites of S-1 (3-9).Cys 707 (SH1) and Cys 697 (SH2) are located on the two different helixes in the C-terminal segment of the S-1 catalytic domain and separated from one another by 19 Å (2). The two helixes are kinked at a conserved glycine residue (Gly 699 ) found in a bend joining two helixes. Recent experiments have led to a conclusion that the fulcrum point for the swinging motion of the lever arm is in the vicinity of the SH1-SH2 region (4 -9). Furthermore, Gly 699 (close to SH2) and Gly 710 (close to SH1) have been proposed to act as pivot points or flexible hinges for such motion (8,10,11). These data show the importance of motion in the SH1-SH2 region in the force generation cycle of myosin. However, the precise conformational changes of this region that are involved in lever arm movement are still unknown.One approach for understanding the involvement of the SH1-SH2 region in lever arm movement is a precise comparison of the individual conformational changes around SH1 and SH2 during ATPase cycle of S-1. Such a study could...

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Defining the “fast-reacting” thiols of myosin by reaction with 1,5 IAEDANS

Cited by 74 publications

References 13 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.

Effect of Complexes of ADP and Phosphate Analogs on the Conformation of the Cys707‐Cys697 Region of Myosin Subfragment 1

ATP-induced Opposite Changes in the Local Environments around Cys697 (SH2) and Cys707 (SH1) of the Myosin Motor Domain Revealed by the Prodan Fluorescence

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