Synthesis and characterization of novel spin-labeled photoaffinity nonnucleoside analogues of ATP as structural and EPR probes for myosin.

Two new spin-labeled photoreactive nonnucleoside ATP analogues, 1-(4-azido-2-nitrophenyl)amino-3-(1-oxyl-2,2,5, 5-tetramethylpyrrolidinyl-3-carbamido)-2-propyl triphosphate (SL-NANTP) and 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6, 6-tetramethyl-4-piperidylidene)di(oxymethylene) ethyl triphosphate (SSL-NANTP), were synthesized and characterized. This study aims to develop a second generation of NANTP-based analogues containing immobile spin labels that can be used to monitor conformational changes in myosin during the contractile cycle of muscle. Previous studies have shown that both a photoaffinity nonnucleoside ATP analogue, 2-[(4-azido-2-nitrophenyl)amino] ethyl triphosphate (NANTP) [Nakamaye et al. (1985) Biochemistry 24, 5226-5235], and a photoaffinity ATP analogue, 3'(2')-O-4-[4-oxo-(4-amino-2,2,6, 6-tetramethyl-piperidino-1-oxyl)-4-benzoyl] benzoyl adenosine 5'-triphosphate (SL-Bz(2)ATP) [Wang et al. (1999) J. Muscle Res. Cell Motil. 20, 743-753], behave like ATP in their interactions with myosin. Remarkably, photolabeled myosin recovers all of its normal enzymatic properties after treatment with actin in the presence of MgATP [Luo et al. (1995) Biochemistry 34, 1978-1987]. For SL-NANTP, the spin label moiety is attached to NANTP via an aminomethyl side chain. In SSL-NANTP, attachment is via a restricted spiro ring. The two new probes interact with myosin subfragment-1 (S1) in a manner analogous to ATP, and after photoincorporation, labeled S1 recovers full activity after treatment with actin and MgATP. The electron paramagnetic resonance (EPR) spectrum resulting from S1 photolabeled with SL-NANTP shows a very high degree of probe mobility. However, the EPR spectrum of S1 photolabeled with SSL-NANTP shows that the probe is highly immobilized with respect to S1, constrained to move within a cone of angle 52 degrees (full-width, half-max). Unlike the parent, NANTP, which photolabels on the 23 kDa tryptic fragment of S1, SSL-NANTP photolabels on the 20 kDa fragment. Its highly immobile nature means that it is potentially a useful reporter group to monitor cross-bridge motion in muscle fibers.

X-ray structures of the Dictyostelium discoideum myosin motor domain with six non-nucleotide analogs.

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with dinitrophenylaminoethyl-, dinitrophenylaminopropyl-, o-nitrophenylaminoethyl-, m-nitrophenylaminoethyl-, p-nitrophenylaminoethyl-, and o-nitrophenyl-N-methyl-aminoethyl-diphosphate.beryllium fluoride have been determined to better than 2.3-A resolution. The structure of the protein and nucleotide binding pocket in these complexes is very similar to that of S1dC.ADP.BeF(x) (Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960-8972). The position of the triphosphate-like moiety is essentially identical in all complexes. Furthermore, the alkyl-amino group plays the same role as the ribose by linking the triphosphate to the adenine binding pocket; however, none of the phenyl groups lie in the same position as adenine in S1dC.MgADP.BeF(x), even though several of these nucleotide analogs are functionally equivalent to ATP. Rather the former location of adenine is occupied by water in the nanolog complexes, and the phenyl groups are organized in a manner that attempts to optimize their hydrogen bonding interactions with this constellation of solvent molecules. A comparison of the kinetic and structural properties of the nanologs relative to ATP suggests that the ability of a substrate to sustain tension and to generate movement correlates with a well defined interaction with the active site water structure observed in S1dC.MgADP.BeF(x).

Fingerprint patterns from laser-induced azido photochemistry of spin-labeled photoaffinity ATP analogs in matrix-assisted laser desorption/ionization mass spectrometry.

The photochemical reaction of azide derivatives induced by ultraviolet (UV) laser in matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is reported. A novel synthesized class of azide aromatic derivatives, spin-labeled photoaffinity non-nucleoside adenosine triphosphate (ATP) analogs which are useful probes in study of muscle contraction mechanism, is used in this investigation. In the negative ion MALDI spectra of these ATP analogs, "fingerprint" peaks corresponding to [M - 10 - 1]-, [M - 12 - 1]-, [M - 16 - 1]-, [M - 26 - 1]-, [M - 28 - 1]-, [M - 41 - 1]-, and [M - 42 - 1]- were observed with relative intensities depending on the MALDI matrix. Only the [M - 16 - 1]- is present in the similar mass spectra of the analog in which the azido group is replaced by a hydrogen. A model is suggested for the photochemical reactions of azide derivatives under UV laser irradiation. The photoreaction fingerprint information is diagnostically useful in characterization of azido compounds, especially for spin-labeled photoaffinity non-nucleoside ATP analogs.

Synthesis of a spin-labeled photoaffinity ATP analogue, and its use to specifically photolabel myosin cross-bridges in skeletal muscle fibers.

A spin-labeled photoaffinity ATP analogue 3'(2')-O-[4-[4-oxo-(4-amido-2,2,6,6-tetramethyl-piperidino-1-oxyl)]-benz oyl]benzoyl adenosine 5'-triphosphate (SL-Bz2ATP) was synthesized and used to photolabel myosin in muscle fibers. Previous work has shown that 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz2ATP) photolabeled Ser-324 of the 50 kDa tryptic fragment of skeletal S1 heavy chain. In this work, [alpha-32P]SL-Bz2ATP was hydrolyzed and trapped as the diphosphate analogue with Co2+ and orthovanadate at the active site of myosin in rabbit psoas muscle fibers. After UV irradiation, the myosin heavy chain was the only protein band found to be significantly photolabeled as assayed by gel electrophoresis and radioactivity counting. The labeling was localized after brief trypsin digestion by SDS-PAGE to be on the 50 kDa tryptic fragment of the S1 heavy chain. Ca. 35% of the myosin in fibers was covalently photolabeled. The fibers photolabeled with SL-Bz2ATP had the same active tension and maximum shortening velocity as the control fibers. The resulting spin label on myosin was too mobile to report the orientation of the heads in fibers. Nonetheless, this is the first work to show the feasibility of utilizing active site binding and photoaffinity labeling to place covalent spectroscopic probes at the myosin active site in fibers with high specificity and yield without affecting mechanical function.

Conformational changes between the active-site and regulatory light chain of myosin as determined by luminescence resonance energy transfer: the effect of nucleotides and actin.

Myosin is thought to generate movement of actin filaments via a conformational change between its light-chain domain and its catalytic domain that is driven by the binding of nucleotides and actin. To monitor this change, we have measured distances between a gizzard regulatory light chain (Cys 108) and the active site (near or at Trp 130) of skeletal myosin subfragment 1 (S1) by using luminescence resonance energy transfer and a photoaffinity ATP-lanthanide analog. The technique allows relatively long distances to be measured, and the label enables site-specific attachment at the active-site with only modest affect on myosin's enzymology. The distance between these sites is 66.8 +/- 2.3 A when the nucleotide is ADP and is unchanged on binding to actin. The distance decreases slightly with ADP-BeF3, (-1.6 +/- 0.3 A) and more significantly with ADP-AlF4 (-4.6 +/- 0.2 A). During steady-state hydrolysis of ATP, the distance is temperature-dependent, becoming shorter as temperature increases and the complex with ADP.Pi is favored over that with ATP. We conclude that the distance between the active site and the light chain varies as Acto-S1-ADP approximately S1-ADP > S1-ADP-BeF3 > S1-ADP-AlF4 approximately S1-ADP-Pi and that S1-ATP > S1-ADP-Pi. The changes in distance are consistent with a substantial rotation of the light-chain binding domain of skeletal S1 between the prepowerstroke state, simulated by S1-ADP-AlF4, and the post-powerstroke state, simulated by acto-S1-ADP.

Raman difference spectroscopic studies of the myosin S1.MgADP.vanadate complex.

The Raman spectra of the nonbridging V--O bonds in the myosin S1.MgADP.Vi complex, often believed to be a transition-state analogue for the phosphotransfer reaction catalyzed by myosin, and in a vanadate solution model compound have been obtained using Raman difference spectroscopic techniques. A symmetric/asymmetric pair of modes at 870 cm-1 is found for vanadate in solution while three bands are found in the myosin S1.MgADP.Vi complex at 870, 844, and 829 cm-1. Using empirical relationships that relate bond order/bond lengths to stretch frequencies, the bond order and bond length of the three nonbridging V--O bonds of vanadate in solution were determined to be 1.43 vu (+/-0.04 vu) and 1.669 A (+/-0.004 A), respectively. The average bond order and bond length of the nonbridging V--O bonds in the S1.MgADP.Vi complex were determined to be 1.38 vu and 1.683 A. A normal-mode analysis suggests that the VO32- moiety approaches a planar conformation in the enzymic complex. Ab initio calculations show that a water molecule at the S1 ATPase binding site, in line with the apical O-V bond in the ADP-Vi moiety and believed to be the attacking nucleophile in the phosphotransfer reaction, can account well for the changes in frequencies of vanadate when it binds to the protein by forming a moderately strong V-O(H2) bond. Hence, an important role determining the ATPase activity at the active site of myosin appears to be a strategic positioning of this in-line water molecule. Assuming that the distortions that vanadate undergoes upon forming the S1.MgADP.Vi complex are analogous to the changes of the gamma-phosphate of ATP in the transition state of the myosin-catalyzed hydrolysis, our results suggest that this reaction proceeds close to a concerted (SN2-like) process.

Chemistry and mechanism of vanadate-promoted photooxidative cleavage of myosin.

Irradiation of the stable myosin subfragment 1(S1).MgADP.orthovanadate (Vi) complex results in oxidation of an active site serine (Ser-180) to a serine aldehyde [Cremo, C. R., Grammer, J. C., & Yount, R. G. (1989) J. Biol. Chem. 264, 6608-6611]. This photomodified S1 will reform a new MgADP.Vi complex and upon a second irradiation, the S1 heavy chain is cleaved into 21 kDa NH2-terminal and 74 kDa COOH-terminal fragments. When S1, in which the side chain of Ser-180 was tritiated, was photocleaved tritium was released from the protein suggesting that cleavage was occurring at Ser-180. The 21 kDa NH2-terminal fragment was resistant to carboxypeptidase digestion, and the 74 kDa COOH-terminal fragment yielded no sequence by Edman degradation, indicating that parts of Ser-180 went to each fragment. To identify these parts, the two cleavage fragments were isolated and chemically (21 kDa) or enzymatically (74 kDa) cleaved, and the resulting peptides were separated by reversed phase HPLC. The peptides immediately down- and up-stream from Ser-180 were isolated and the blocking groups were identified by mass spectrometry. The 21 kDa fragment peptide was blocked with a carboxamide on Glu-179 (confirmed by HPLC and capillary electrophoresis in comparison with peptide standards), while the NH2 group of Gly-181 of the 74 kDa fragment was blocked with an oxalyl group (verified by enzymatic analysis for oxalate). The side chain of Ser-180 was released as formate. O2 is required for photocleavage. Cleavage experiments in the presence of 18O2 showed one atom of 18O labeled the oxalyl group. A mechanism in which O2 adds to a free radical on the alpha-carbon of Ser-180 with a subsequent Criegee type rearrangement is proposed to explain both the kinetics and products of the photocleavage.

The active site of myosin.

The significance of myosin has been expanded recently with the realization that it is found in every eukaryotic cell, where it has a role in cytokinesis, cell division, and vesicle transport. Advances in molecular genetics and expression systems related to myosin and actin have helped to reveal the extent of the myosin superfamily. New motility assays and techniques have provided information about the residues involved in ATP hydrolysis and the conformational change induced by nucleotide binding. The results of these techniques revealing structural and functional information combined with previous studies of the active site of myosin should provide future direction for studying this exciting and rapidly moving area of biochemistry.

Is myosin a "back door" enzyme?

ATP has been modeled into the active site of chicken skeletal myosin subfragment-1 using the adenylate kinase.Ap5A structure as a starting reference. The resulting docked ATP.S1 structure is justified in that it rationalizes the photolabeling data from several ATP analogs. The gamma-phosphate of ATP sits at the bottom of the active site pocket and is partially visible via a view along the prominent 50-kDa cleft of S1 but not when viewed from above the active site. It is postulated that actin binding promotes the movement of the P-loop and Arg-245 to allow Pi from ATP to leave via a "back-door" in the 50-kDa fragment while ADP is still bound at the active site. Such a mechanism can explain a number of experimental observations, including the kinetics of ATP hydrolysis, the nucleotide dependence of Pi exchange into ATP, and the formation of stable myosin.ADP.vanadate complexes in muscle fibers.

Photoaffinity ADP analogs as covalently attached reporter groups of the active site of myosin subfragment 1.

The enzymatic properties of rabbit skeletal myosin subfragment 1 (S1) have been determined after photoaffinity labeling the active site with two ADP analogs. These analogs, 2-[(4-azido-2-nitrophenyl)-amino]ethyl diphosphate (NANDP) and the fluorescent analog 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine diphosphate (Bz2 epsilon ADP), label the heavy chain residues Trp 130 and Ser-324, respectively. These residues in the crystal structure of chicken skeletal S1 are on either side of the entrance to the active site pocket (Rayment et al., 1993b). Here S1 was photolabeled with NANDP or Bz2 epsilon ADP after trapping with vanadate (Vi). Both of the photolabeled S1 preparations had normal MgATPase activities after removal of vanadate by actin treatment. These results show that the covalently tethered nucleotide analogs could move out of the active site and be replaced by MgATP. Experiments that monitored the fluorescence emission intensity, polarization, and quenching by acrylamide of S1 photolabeled with Bz2 epsilon ADP show that the covalently linked analog was displaced out of the active site cleft by MgATP (or MgATP and actin) but not by ATP in the absence of Mg2+ ions. The effective concentration of the tethered ethenoadenosine diphosphate at the active site, determined by competition with MgATP, was calculated to be 10 mM. In the absence of Mg2+ ions, ATP was unable to compete with the bound analog. Binding constants of the S1 photolabeled with Bz2 epsilon ADP to actin were 1.5 x 10(5) and 5.8 x 10(5) M-1 at 200 and 20 mM ionic strength, respectively, showing that actin binding affinities are similar to those obtained for S1.ADP. The binding of actin in the absence of MgATP did not produce any change in the emission intensity, polarization, or quenching by acrylamide of the tethered ethenoadenosine diphosphate, indicating that the conformation of the pocket around the adenine ring was unchanged. However, the binding of actin did destabilize Vi, which had been previously trapped in the form of photolabeled S1-Vi complexes. These results indicate that actin binding primarily affects the gamma-phosphate binding site but not the adenine ring binding site.

Photoaffinity labeling of skeletal myosin with 2-azidoadenosine triphosphate.

The purine binding site of ATP on skeletal muscle myosin has been photoaffinity labeled with 2-azidoadenosine diphosphate (2-N3ADP). 2-N3ADP was stably trapped at the active site (t1/2 approximately 5 days, 0 degree C) by complexation of the two heavy chain reactive thiols (Cys-697 and Cys-707) with Co(III)phenanthroline. Photoincorporation occurred only in the 23-kDa NH2-terminal tryptic fragment of the heavy chain. Extensive serial digestion of photolabeled subfragment 1 of myosin by trypsin and subtilisin yielded a series of labeled peptides which were purified by HPLC. Sequence and radiolabeling analysis of eight photolabeled peptides all indicated that tryptophan-130 was the only labeled residue. This site of labeling confirms earlier photolabeling studies with the non-nucleotide ADP analogue, 2[(4-azido-2-nitrophenyl)-amino]ethyl diphosphate (NANDP), which also labeled Trp-130 [Okamoto, Y., & Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1579]. Comparison of the structures of 2-N3ADP and NANDP indicate that their azido groups can be superimposed if both analogues bind to the active site in an extended conformation in a manner analogous to the anti conformation of ATP.

Photolabeling evidence for calcium-induced conformational changes at the ATP binding site of scallop myosin.

A change in the conformation of the active site of scallop myosin under the influence of regulatory amounts of Ca2+ has been identified by use of the ADP photoaffinity analog 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate (NANDP). NANDP, trapped at the active site with Mn2+ and vanadate, photolabeled preferentially Arg-128 of the heavy chain in the absence of added Mg2+ and Ca2+ [Kerwin, B. & Yount, R. (1992) Bioconjugate Chem. 3, 328-336]. However, addition of 2 mM Mg2+ and regulatory amounts of Ca2+ (0.01-1 microM) shifted the predominant labeling to Cys-198 of the heavy chain in a Ca(2+)-dependent manner. This Ca(2+)-dependent change in the photolabeling pattern was absent when the regulatory light chains were removed or when the unregulated head (subfragment 1) was examined under similar conditions. These results demonstrate that both Arg-128 and Cys-198 are part of the purine binding site which undergoes a conformational change in response to Ca2+ binding to the regulatory domain.

Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine.

The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity labeling of scallop myosin with 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate: identification of an active site arginine analogous to tryptophan-130 in skeletal muscle myosin.

The ADP photoaffinity analogue 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate (NANDP) was used to photolabel the ATP binding site of scallop myosin. Approximately 1 mol of NANDP per mol of myosin was trapped at the active site by complexation with vanadate and manganese. ADP, but not AMP, inhibited trapping of NANDP. The trapped NANDP photolabeled up to 37% of the myosin upon UV irradiation. Papain subfragment-1 prepared from the photolabeled myosin was digested with trypsin, and the major photolabeled tryptic peptides were isolated by reversed-phase HPLC. The amino acid sequence of the major labeled peptide was X-Leu-Pro-Ile-Tyr-Thr-Asp-Ser-Val-Ile-Ala-Lys, where X represents the photolabeled amino acid Arg128. Previously, Trp130 of rabbit skeletal muscle myosin has been shown to be photolabeled by NANDP [Okamoto, Y., and Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1580]. Scallop and rabbit skeletal muscle myosin display a high degree of sequence similarity in this region with Arg128 in an equivalent position as Trp130. These results suggest that the composition of the purine binding site is analogous in both myosins and that Arg and Trp play a similar role in binding ATP, despite the marked differences of their side chains.

Photochemical mapping of the active site of myosin.

The active sites of myosin from skeletal, smooth and scallop muscle have been partly characterized by use of a series of photoreactive analogues of ATP. Specific labelling was attained by trapping these analogues in their diphosphate forms at the active sites by either cross-linking two reactive thiols (skeletal myosin) or by formation of stable vanadate-metal ion transition state-like complexes (smooth muscle and scallop myosin). By use of this approach combined with appropriate chemistry, several key residues in all three myosins have been identified which bind at or near the adenine ring, the ribose ring and to the gamma-phosphate of ATP. This information should aid in the solution of the crystal structure of the heads of myosin and in defining a detailed structure of the ATP binding site.

Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate.

The active-site topology of smooth muscle myosin has been investigated by direct photoaffinity-labeling studies with [3H]ADP. Addition of vanadate (Vi) and Co2+ enabled [3H]ADP to be stably trapped at the active site (t1/2 greater than 5 days at 0 degrees C). The extraordinary stability of the myosin.Co2+.[3H]ADP.Vi complex allowed it to be purified free of excess [3H]ADP before irradiation began and ensured that only active-site residues became labeled. Following UV irradiation, approximately 10% of the trapped [3H]ADP became covalently attached at the active site. All of the [3H]ADP incorporated into the 200-kDa heavy chain, confirming earlier results using untrapped [alpha-32P]ATP [Maruta, H., & Korn, E. (1981) J. Biol. Chem. 256, 499-502]. After extensive trypsin digestion of labeled subfragment 1, HPLC separation methods combined with alkaline phosphatase treatment allowed two labeled peptides to be isolated. Sequence analysis of both labeled peptides indicated that Glu-185 was the labeled residue. Since Glu-185 has been previously identified as a residue at the active site of smooth myosin using [3H]UDP as a photolabel [Garabedian, T. E., & Yount, R. G. (1990) J. Biol. Chem. 265, 22547-22553], these results provide further evidence that Glu-185, located immediately adjacent to the glycine-rich loop, is located in the purine binding pocket of the active site of smooth muscle myosin.

Mechanics of glycerinated muscle fibers using nonnucleoside triphosphate substrates.

We have investigated the ability of the photoaffinity, nonnucleotide ATP analogues, 2-[(4-azido-2-nitrophenyl) amino] ethyl triphosphate (NANTP) and 2-[(4-azido-2-nitrophenyl) amino] propyl triphosphate (PrNANTP), to support active contraction in glycerinated rabbit psoas fibers. At millimolar concentrations, in the absence of calcium, both analogues relaxed fibers. In the presence of calcium, MgNANTP produced isometric tension and stiffness that were one-half to two-thirds the values obtained in MgATP. Maximum shortening velocity and the calcium-activated, myofibrillar catalyzed rate of hydrolysis were approximately the same for MgNANTP as for MgATP. With MgNANTP as the substrate, increasing concentrations of the diphosphate analogue, MgNANDP, inhibited shortening velocity but did not change isometric tension. The addition of increased concentrations of orthophosphate (P) decreased tension while shortening velocity increased. Thus, the effects of the hydrolysis products of NANTP were quite similar to those observed previously for ADP and P in the presence of MgATP. Taken together, these observations show that MgNANTP binds to, and functions in the active site of myosin in a manner quite analogous to MgATP. Thus, the aryl azido group should serve as a valid photoaffinity label for the purine portion of the active site. In contrast, MgPrNANTP, which differs from MgNANTP only in an extra CH2 spacer between the nitrophenyl ring and the triphosphate moiety did not support isometric tension or active shortening in the presence of calcium. Fiber stiffness increased in the presence of calcium and MgPrNANTP, with a calcium-activated, myofibrillar MgPrNANTPase which was about half that obtained with MgATP. Thus, in the presence of MgPrNANTP, cross-bridges appeared to be cycling through states that were attached to actin, but not producing force.

Photolabeling of the 6 and 10 S conformations of gizzard myosin with 3'(2')-O-(4-Benzoyl)benzoyl-ATP. Proline 324 is near the active site.

3'(2')-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP) was used as a photoaffinity label of the ATP binding site of unphosphorylated chicken gizzard myosin. Specific photolabeling of the active site of 6 S myosin was assured by forming a stable myosin.Co(II)Bz2ADP.orthovanadate complex (termed trapping) prior to irradiation. Co2+ was used in place of Mg2+ to prevent the known photoreaction of vanadate with myosin which destabilizes the trapped complex. [3H] Bz2ADP.Pi was also stably trapped on gizzard myosin by forming the 10 S folded conformation of the protein in the presence of [3H]Bz2ATP and Mg2+. Irradiation of 6 S myosin containing orthovanadate trapped [3H] Bz2ADP or 10 S trapped [3H]Bz2ADP.Pi gave 32 and 30% covalent incorporation, respectively. The 50-kDa and precursor 68-kDa tryptic peptides of the subfragment-1 heavy chain derived from both forms of myosin were found to contain essentially all of the covalently attached [3H]Bz2ADP. Parallel experiments with untrapped [3H]Bz2ADP showed extensive nonspecific labeling of all of the major tryptic peptides and the light chains. Eight labeled peptides, isolated from 6 and 10 S photolabeled myosin, contained the sequence G319-H-V-P-I-X-A-Q326, where X corresponds to labeled proline 324. [14C]Bz2ADP was previously shown to label serine 324 in skeletal subfragment-1 (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995), which corresponds to alanine 325 in the gizzard sequence. Thus, this region of the 50-kDa tryptic fragment, near the nucleotide binding site, in both skeletal and smooth muscle myosins, must fold in essentially the same manner.