Photoaffinity labelling with an ATP analog of the N-terminal peptide of myosin

Szilágyi, László; Bálint, M.; Sreter, F. A.; Gergely, J.

doi:10.1016/0006-291x(79)92047-3

Cited by 122 publications

(57 citation statements)

References 10 publications

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“…Since the azidonitrobenzoyl group attached to the ribose ring of ADP spans only several Angstroms, it is very likely that a part of the Mr 25,000 segment participates in ATP binding. This result is consistent with the previous observation that a photoreactive ATP analog, arylazido-(3alanyl ATP (20), affinity-labeled the Mr 25,000 segment (12). Participation of the segment in the ATPase site was also demonstrated by another ADP analog, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (14).…”

Section: Discussionsupporting

confidence: 92%

“…Locations of the actin-head interface were identified along polypeptide chains of actin as well as S1 by the use of chemical crosslinkings (8)(9)(10)(11). As for the ATPase site, several groups tried to map its location along polypeptides of myosin by affinity labeling with photoreactive ATP analogs (12)(13)(14). These studies showed that part of the Mr 25,000 segment and part of the Mr 50,000 segment of S1 heavy chain were in proximity of the ATPase site.…”

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confidence: 99%

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Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog.

Sutoh¹,

Yamamoto²,

Wakabayashi³

1986

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

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An ADP analog carrying a biotin moiety and a photoreactive group was synthesized. In the presence of vanadate ion (VI), the analog was tightly trapped into the ATPase site of heavy meromyosin (HMM) or myosin subfragment 1 (S1) in an ADP analog/ATPase site molar ratio of 1:1. UV illumination on the HMM (or Sl)-V--ADP analog complex resulted in covalent incorporation ofthe analog into the ATPase site. About 15% ofthe trapped analog was crosslinked to HMM or S1. Mapping of the crosslinking site of the analog showed that the N-terminal Mr 25,000 segment of the heavy chain participated in binding the ADP analog. The biotin moiety of the analog covalently incorporated into the ATPase site was visualized in electron microscopy by attaching an avidin oligomer. Rotary-shadowed images of the HMM-avidin complex revealed that the crosslinked ADP analog was located about 140 A from the head-rod junction on the head. The result indicates that the ATPase site of myosin is about 140 A apart from the head-rod junction along the head.Myosin head has two essential sites for force generation in muscle: one is the ATPase site and the other is the actinbinding site. It is of vital importance to identify spatial locations of these sites on the head and also to locate these sites along polypeptides of the head in order to understand muscle contraction at the molecular level.As for the actin-binding site, three-dimensional reconstitution of electron microscopic images of the rigor complex of actin and myosin head (subfragment 1 or S1) showed how the head binds to F-actin (1-7). Locations of the actin-head interface were identified along polypeptide chains of actin as well as S1 by the use of chemical crosslinkings (8-11). As for the ATPase site, several groups tried to map its location along polypeptides of myosin by affinity labeling with photoreactive ATP analogs (12)(13)(14). These studies showed that part of the Mr 25,000 segment and part of the Mr 50,000 segment of S1 heavy chain were in proximity of the ATPase site. Spatial location of the site was investigated by a fluorescence energy-transfer experiment (15), which showed that the relative distance between the ATPase site and one ofthe most reactive thiols of myosin (SH1) was about 40 A. Since we have located the SH1 thiol about 130 A from the head-rod junction on the myosin head by electron microscopy with a recently developed avidin-biotin system (16), it seems that the ATPase site of myosin is in the distal half of the head.In this study, we tried to identify spatial location of the ATPase site by electron microscopy. For the purpose, we synthesized a novel photoreactive ADP analog carrying a biotin moiety for use in affinity labeling. After photoaffmity labeling of the ATPase site with the analog, the biotin moiety was visualized by attaching avidin (16). Analysis of rotaryshadowed images indicates that the site is located about 140 A apart from the head-rod junction on the head. MATERIALS AND METHODSSynthesis of a Biotinylated Photoreactive Analog of ADP for Affinit...

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Section: Discussionsupporting

confidence: 92%

mentioning

confidence: 99%

Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog.

Sutoh¹,

Yamamoto²,

Wakabayashi³

1986

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

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“…These structural observations imply that R128 in scallop or its equivalent in skeletal muscle myosin is involved in ATP recruitment to the nucleotide-binding pocket. Evidence for this role of R128 is provided by cross-linking studies using photoaffinity ATP analogs (28)(29)(30). Moreover, in isoforms lacking a tight contact between the pair of residues equivalent to R128͞E184, ATP binding is weaker by one or two orders of magnitude.…”

Section: Resultsmentioning

confidence: 99%

Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding

Risal

Gourinath

Himmel

et al. 2004

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

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Structural studies of myosin have indicated some of the conformational changes that occur in this protein during the contractile cycle, and we have now observed a conformational change in a bound nucleotide as well. The 3.1-Å x-ray structure of the scallop myosin head domain (subfragment 1) in the ADP-bound near-rigor state (lever arm Ϸ45°to the helical actin axis) shows the diphosphate moiety positioned on the surface of the nucleotide-binding pocket, rather than deep within it as had been observed previously. This conformation strongly suggests a specific mode of entry and exit of the nucleotide from the nucleotide-binding pocket through the so-called ''front door.'' In addition, using a variety of scallop structures, including a relatively high-resolution 2.75-Å nucleotide-free near-rigor structure, we have identified a conserved complex salt bridge connecting the 50-kDa upper and N-terminal subdomains. This salt bridge is present only in crystal structures of muscle myosin isoforms that exhibit a strong reciprocal relationship (also known as coupling) between actin and nucleotide affinity.M yosin is a motor protein that transduces ATP hydrolysis into mechanical work, leading to its translocation along filamentous actin. It is believed that the small conformational changes induced by enzymatic activity in the myosin ''motor domain'' (MD) are amplified by the motion of the ''lever arm.'' Three weak actin-binding (actin-detached) states have been identified crystallographically for scallop myosin subfragment 1 (S1) (1). These and other structures reveal that the motor function of myosin S1 is coordinated by three subdomains (50-kDa upper and lower subdomains and the converter) that rotate as rigid bodies around the relatively stable N-terminal subdomain (1-3). These rotations depend on conformational changes in three flexible joints (switch II, relay, and the SH1 helix) between the subdomains. Three-dimensional reconstructions of electron microscopic images of actin decorated by S1 of several myosin isoforms (4-6) have led to the suggestion that actin binds to portions of the 50-kDa upper and lower subdomains, whereas various crystal structures (see, for example, refs. 3 and 7-9) indicate that ATP binds at the opposite side of the myosin head in a pocket between the 50-kDa upper and Nterminal subdomains (Figs. 1 and 2A).One of the three structural conformations, the so-called ''prepower stroke,'' corresponds biochemically to the ADP⅐P i transition state (1,8,(10)(11)(12). Here, S1 displays a primed lever arm (Ϸ90°to the actin filament axis), closure of the 50-kDa cleft's base (which is near the nucleotide-binding pocket), and a bent switch II that interacts with both the nucleotide and switch I (a loop in the 50-kDa upper subdomain that coordinates the nucleotide). After this state, the tight binding of myosin to actin results in the so-called ''power stroke'' (after which the lever arm is Ϸ45°to the actin filament), a process believed to be initiated by the near-complete closing of the 50-kDa cleft (4-6). This...

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“…The 75-kDa fragment is further degraded into an N-terminal 25-kDa fragment and an adjacent 50-kDa polypeptide with the transient appearance of a 27-kDa fragment [I -31. It has been suggested that the 25-kDa fragment, which was found to be the binding site of a photoaffinity ATP analog [4], is formed by two parallel routes directly from the 75-kDa fragment and indirectly with the 27-kDa fragment as precursor [ 5 ] .Alterations in the proteolytic fragmentation pattern induced by actin, nucleotide or metal binding indicate that structural changes take place in the myosin head region upon their interactions. Thus actin inhibits the proteolytic cleavage of the S1 heavy chain at the junction between the 50-kDa and 20-kDa fragments.…”

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confidence: 99%

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Effect of nucleotides, divalent cations and temperature on the tryptic susceptibility of myosin subfragment 1

Mócz

Szilágyi

et al. 1984

European Journal of Biochemistry

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The kinetics of tryptic breakdown of the heavy chain of chymotryptic myosin subfragment 1 (Sl) according to the following scheme (where the numbers respresent approximate masses in kDa) are altered at 21 "C by divalent process affects the tryptic fragmentation of S1 similarly to, but less effectively than, nucleotides. Acts-S1 formation prevents the effect of ATP on fragmentation. At 37 'C S1 loses ATPase activity; tryptic digestion proceeds more rapidly and the 50-kDa and 25-kDa fragments are degraded to small peptides. Nucleotides protect against the effects of higher temperature by producing conformational changes not only in the 27-kDa N-terminal portion (containing the putative nucleotide binding site) of the heavy chain of S1 but also in the 50-kDa peptide.Previous studies have shown that, on further digestion with trypsin, the heavy chain of chymotryptic myosin subfragment 1 (Sl) is split into two fragments with approximate masses of 75 kDa and 20kDa; the latter contains the two reactive thiols, SH-1 and SH-2 [I]. The 75-kDa fragment is further degraded into an N-terminal 25-kDa fragment and an adjacent 50-kDa polypeptide with the transient appearance of a 27-kDa fragment [I -31. It has been suggested that the 25-kDa fragment, which was found to be the binding site of a photoaffinity ATP analog [4], is formed by two parallel routes directly from the 75-kDa fragment and indirectly with the 27-kDa fragment as precursor [ 5 ] .Alterations in the proteolytic fragmentation pattern induced by actin, nucleotide or metal binding indicate that structural changes take place in the myosin head region upon their interactions. Thus actin inhibits the proteolytic cleavage of the S1 heavy chain at the junction between the 50-kDa and 20-kDa fragments. The cleavage abolishes the A preliminary report has been presented [(1982) Biophys. J . 37, 38al.Abbreviations. AdoPP[NH]P, adenosine 5'-[P,y-imido]triphosphate; HMM, heavy meromyosin; S1, myosin subfragment 1 ; Nbs,, 5,5'-dithiobis(2-nitrobenzoic acid) ; NaDodSO,, sodium dodcecyl sulfate.

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Photoaffinity labelling with an ATP analog of the N-terminal peptide of myosin

Cited by 122 publications

References 10 publications

Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog.

Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog.

Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding

Effect of nucleotides, divalent cations and temperature on the tryptic susceptibility of myosin subfragment 1

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