Phosphorylation-dependent Structural Changes in the Regulatory Light Chain Domain of Smooth Muscle Heavy Meromyosin

Abstract

Smooth muscle heavy meromyosin, a double-headed proteolytic fragment of myosin lacking the COOH-terminal two-thirds of the tail, has been shown previously to be regulated by phosphorylation. To examine phosphorylation-dependent structural changes near the head-tail junction, we prepared five well regulated heavy meromyosins containing single-cysteine mutants of the human smooth muscle regulatory light chain labeled with the photocross-linking reagent, benzophenone-iodoacetamide. For those mutants that generated cross-links, only one type of cross-linked species was observed, a regulatory light chain dimer. Irradiated mutants fell into two classes. First, for Q15C, A23C, and wild type (Cys-108), a regulatory light chain dimer was formed for dephosphorylated but not thiophosphorylated heavy meromyosin. These data provide direct chemical evidence that in the dephosphorylated state, Gln-15, Ala-23, and Cys-108 on one head are positioned near (within 8.9 Å) the regulatory light chain of the partner head and that thiophosphorylation abolishes proximity. This behavior was also observed for the Q15C mutant on a truncated heavy meromyosin lacking both catalytic domains. For the actin-heavy meromyosin complex, cross-links were formed in both de- and thiophosphorylated states. S59C and T134C mutants were in a second mutant class, where regulatory light chain dimers were not detected in dephosphorylated or thiophosphorylated heavy meromyosin, suggesting positions outside the region of interaction of the regulatory light chains. Smooth muscle heavy meromyosin, a double-headed proteolytic fragment of myosin lacking the COOH-terminal two-thirds of the tail, has been shown previously to be regulated by phosphorylation. To examine phosphorylation-dependent structural changes near the head-tail junction, we prepared five well regulated heavy meromyosins containing single-cysteine mutants of the human smooth muscle regulatory light chain labeled with the photocross-linking reagent, benzophenone-iodoacetamide. For those mutants that generated cross-links, only one type of cross-linked species was observed, a regulatory light chain dimer. Irradiated mutants fell into two classes. First, for Q15C, A23C, and wild type (Cys-108), a regulatory light chain dimer was formed for dephosphorylated but not thiophosphorylated heavy meromyosin. These data provide direct chemical evidence that in the dephosphorylated state, Gln-15, Ala-23, and Cys-108 on one head are positioned near (within 8.9 Å) the regulatory light chain of the partner head and that thiophosphorylation abolishes proximity. This behavior was also observed for the Q15C mutant on a truncated heavy meromyosin lacking both catalytic domains. For the actin-heavy meromyosin complex, cross-links were formed in both de- and thiophosphorylated states. S59C and T134C mutants were in a second mutant class, where regulatory light chain dimers were not detected in dephosphorylated or thiophosphorylated heavy meromyosin, suggesting positions outside the region of interaction of the regulatory light chains. The actin-activated ATPase activity and motor properties of smooth muscle and nonmuscle myosins are regulated by phosphorylation of the regulatory light chain (1Hartshorne D.J. Johnson L.R. Biochemistry of the Contractile Process in Smooth Muscle: Physiology of the Gastrointestinal Tract. 2nd Ed. Raven Press, New York1987: 423-481Google Scholar, 2Sellers J.R. Curr. Opin. Cell Biol. 1991; 3: 98-104Crossref PubMed Scopus (173) Google Scholar, 3Sellers J.R. Goodson H. Protein Profile. 1995; 2: 1323-1423PubMed Google Scholar). The dephosphorylated forms of these regulated myosins have low ATPase activity and are unable to move actin filaments, whereas phosphorylated forms are activated in both respects. Domain requirements for regulation have been elucidated through studies of various proteolytic and expressed subfragments of SMM. 1The abbreviations SMMsmooth muscle myosinHMMheavy meromyosinRLCregulatory light chainELCessential light chainBP-RLCRLC labeled on cysteine with benzophenone-4-iodoacetamidehisRLCHis6-tagged RLCWTwild typeMOPS4-morpholinepropanesulfonic acidDTTdithiothreitolHMM/BP-RLCHMM exchanged with benzophenone-4-iodoacetamide-labeled RLC (mutant not specified) SMM contains two head domains (S1) attached to a long α-helical coiled-coil domain (tail). Single-headed myosin (4Cremo C.R. Sellers J.R. Facemyer K.C. J. Biol. Chem. 1995; 270: 2171-2175Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and S1 (5Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar, 6Seidel J.C. J. Biol. Chem. 1980; 255: 4355-4361Abstract Full Text PDF PubMed Google Scholar) are active in both dephosphorylated and phosphorylated states. HMM that lacks the COOH-terminal two-thirds of the tail is double-headed and well regulated (5Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar, 7Sellers J.R. J. Biol. Chem. 1985; 260: 15815-15819Abstract Full Text PDF PubMed Google Scholar), but expressed HMMs with shorter tails failed to form double-headed structures and were found to be unregulated (8Matsuura M. Ikebe M. FEBS Lett. 1995; 363: 246-250Crossref PubMed Scopus (30) Google Scholar, 9Sata M. Matsuura M. Ikebe M. Biochemistry. 1996; 35: 11113-11118Crossref PubMed Scopus (47) Google Scholar) as in S1 and single-headed myosin. Therefore, two heads are critical for down-regulation, suggesting that head-head interaction is an important feature of the dephosphorylated state. However, a double-headed structure may not be sufficient for full regulation, and interactions between heads and rods may also be required (10Trybus K.M. Freyzon Y. Faust L.Z. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 48-52Crossref PubMed Scopus (130) Google Scholar). smooth muscle myosin heavy meromyosin regulatory light chain essential light chain RLC labeled on cysteine with benzophenone-4-iodoacetamide His6-tagged RLC wild type 4-morpholinepropanesulfonic acid dithiothreitol HMM exchanged with benzophenone-4-iodoacetamide-labeled RLC (mutant not specified) The general location of the phosphorylated subunit (RLC) at the junction between the heads and tail (11Flicker P.F. Wallimann T. Vibert P. J. Mol. Biol. 1983; 169: 723-741Crossref PubMed Scopus (94) Google Scholar) suggests that RLC-RLC interactions may be a logical consequence of head-head interactions. Indeed, the RLC has been shown to be critical to the regulatory mechanism. In particular, disruption of the COOH-terminal domain (12Ikebe M. Reardon S. Mitani Y. Kamisoyama H. Matsuura M. Ikebe R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9096-9100Crossref PubMed Scopus (33) Google Scholar,13Rowe T. Kendrick-Jones J. EMBO J. 1993; 12: 4877-4884Crossref PubMed Scopus (14) Google Scholar), the central helix (14Ikebe M. Kambara T. Stafford W.F. Sata M. Katayama E. Ikebe R. J. Biol. Chem. 1998; 273: 17702-17707Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), and portions of the NH2-terminal charged region (15Ikebe M. Ikebe R. Kamisoyama H. Reardon S. Schwonek J.P. Sanders II, C.R. Matsuura M. J. Biol. Chem. 1994; 269: 28173-28180Abstract Full Text PDF PubMed Google Scholar) of the RLC alters regulation. Smooth muscle myosin with exchanged skeletal RLC was not activated by phosphorylation (16Trybus K.M. Chatman T.A. J. Biol. Chem. 1993; 268: 4412-4419Abstract Full Text PDF PubMed Google Scholar). In contrast to the RLC, the ELC appears to be less important to regulation because it can be replaced by the skeletal isoform or removed entirely with little effect on regulation (17Trybus K.M. J. Biol. Chem. 1994; 269: 20819-20822Abstract Full Text PDF PubMed Google Scholar). The requirements for full regulation in smooth muscle myosin are complex, because of a subtle interplay between the light chain domain, the actin binding loop (18Rovner A.S. Freyzon Y. Trybus K.M. J. Biol. Chem. 1995; 270: 30260-30263Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), the catalytic domain (19Sata M. Stafford W.F. Mabuchi K. Ikebe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 91-96Crossref PubMed Scopus (30) Google Scholar), and the heavy chain sequence within the light chain domain (20Trybus K.M. Naroditskaya V. Sweeney H.L. J. Biol. Chem. 1998; 273: 18423-18428Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The exact location of the phosphorylated serine (S19 in chicken gizzard) of the RLC is not known. The analogous residue was found to be within a disordered region of the NH2 terminus in the crystal structures of both skeletal S1 (21Rayment I. Rypniewski W.R. Schmidt-Base K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 1-58Google Scholar) and the light chain domain of scallop myosin (22Xie X. Harrison D.H. Schlichting I. Sweet R.M. Kalabokis V.N. Szent-Gyorgyi A.G. Cohen C. Science. 1994; 368: 306-312Google Scholar). It is possible that this region adopts an ordered structure in a double-headed molecule. Clearly, more information about the structure of the head-tail junction including the RLC is needed to understand the phosphorylation-dependent regulatory mechanism. Our approach has been to use direct chemical methods to further define this region by looking for conformational differences between the dephosphorylated and phosphorylated forms of smooth muscle HMM. Specifically, we have placed photocross-linking probes selectively throughout the RLC by use of a set of single cysteine mutants. We have labeled two residues within the putative flexible NH2-terminal region (Q15C and A23C), two within the COOH-terminal domain (Cys-108 and T134C), and one within the NH2-terminal domain (S59C). After exchange of the labeled RLC into HMM, we found that cross-linked RLC-RLC dimers were formed after irradiation of Q15C, A23C, and WT (Cys-108) but not of S59C or T134C. These RLC-RLC dimers were not formed after irradiation of the respective thiophosphorylated HMMs. Our results suggest that regions of the RLC of one head are proximal to the RLC of the other head in dephosphorylated HMM, and this proximity is abolished in the phosphorylated state. We also show that this conformational change occurs independently of the catalytic domain. SMM was prepared from frozen chicken gizzards (23Ikebe M. Hartshorne D.J. J. Biol. Chem. 1985; 260: 13146-13153Abstract Full Text PDF PubMed Google Scholar) from Smooth muscle HMM was from a of SMM as previously (5Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar) a was or A. J. Biol. Chem. 261: Full Text PDF PubMed Google Scholar) that HMM was SMM and HMM were the HMM, nonmuscle heavy meromyosin a isoform lacking both catalytic was a from J. Sellers and The light chain mutants were prepared by a cysteine residue into the human smooth muscle RLC and chain smooth muscle RLC mutants Q15C, A23C, and T134C were prepared by of Gln-15, Ala-23, and with and Cys-108 of was replaced by The for the human RLC was into at the the was to the Cys-108 with and Ala-23, or with The for the Cys-108 to was The for A23C, and T134C mutants were and chain was to the mutant The a at the as well as the to The sequence was The in chain was to the of the region of the RLC with an at the of the The was was the chain with the RLC were by and into the to the NH2 terminus of of the for the were of at was to of was to the to for were by at for and of was to the to for of and on for The was with of of and of of and at for The was at at for The was at with for and at for The was with for and for and were more The RLC mutant was with in the and the was The was to and and labeled with benzophenone-4-iodoacetamide as previously Sellers J.R. C.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The of labeled were the of Sellers J.R. C.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of RLC of HMM for RLC were as previously K.C. C.R. Chem. 3: PubMed Scopus Google Scholar). RLC in the exchange of was removed by a and was between the exchanged HMM and the were and with of and to was and the was on for of that of were removed from the RLC by was to and after was phosphorylated by in myosin light chain PubMed Scopus (47) Google Scholar), and at and for by on thiophosphorylation was by (4Cremo C.R. Sellers J.R. Facemyer K.C. J. Biol. Chem. 1995; 270: 2171-2175Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). were prepared by and to the other for The was to irradiation by were at for and the were through a The two and were sufficient for HMM, as by A. J. Biol. Chem. 261: Full Text PDF PubMed Google Scholar). was to to the To effect of HMM in a was through a with a on as previously Sellers J.R. C.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). was in the of with a from were with were to for were by J. T. 2nd Ed. Scholar) for both RLC and ELC and labeled with were as the and second for RLC from K. and labeled with were as the and second for ELC was to the with RLC and and were with a for a HMM in this HMM was prepared from chicken myosin mutant RLC was with HMM HMM and exchanged for RLC by a previously shown (16Trybus K.M. Chatman T.A. J. Biol. Chem. 1993; 268: 4412-4419Abstract Full Text PDF PubMed Google Scholar, Sellers J.R. C.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, R. Ikebe M. Biochemistry. 1991; PubMed Scopus (20) Google Scholar) to phosphorylation-dependent regulation RLC was removed by is be by the ELC to The of the was in an to be the were removed by to of regulatory properties of HMM. mutant RLC the RLC because of an of the light chain region by that the of RLC both the mutant and the to ELC for the exchanged was within of the for the HMM not This suggests that the of the RLC the exchange The shown in was to mutants and the respective was for ATPase and irradiation ATPase was HMM. The were exchanged with the RLC mutant labeled with were or to the thiophosphorylated thiophosphorylation was in the of and were or to the thiophosphorylated thiophosphorylation was in the of and were or to the thiophosphorylated thiophosphorylation was in the of and are of The of the was within of HMM and mutants were at in at and The of of was J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar). The for dephosphorylated HMMs were at and HMMs were at and The was HMM. The were exchanged with the RLC mutant labeled with were or to the thiophosphorylated thiophosphorylation was in the of and are of The of the was within in a of HMM and mutants were at in at and The of of was J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar). The for dephosphorylated HMMs were at and HMMs were at and The of thiophosphorylation the actin-activated ATPase activity of HMM and mutants are shown in I. to the HMM. from studies (5Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar), the HMMs by thiophosphorylation in the of actin but in the of actin an in activity The exact in the of actin because of thiophosphorylation was to from these data because the dephosphorylated were low and to of However, the of the thiophosphorylated were within a of of the These results show that the were regulated by thiophosphorylation and were for irradiation the effect of irradiation of HMM exchanged with benzophenone-4-iodoacetamide-labeled Q15C RLC The is for mutants. with light to the for to proximal In the dephosphorylated HMM irradiation generated a between and not and actin with the behavior of a RLC-RLC dimer This was as after with RLC but not ELC The RLC-RLC dimer was also generated in the of and and and in the of actin The of RLC to the RLC-RLC dimer was by in the light chain region the ELC for of This that the was about for irradiation including in the of and and The RLC-RLC dimer was the only observed after irradiation was not observed from the RLC to the heavy chain to the In RLC was not To for of RLC were to the positions on the of cross-linked the RLC not However, these were not in with the RLC to the heavy chain in the HMM Therefore, we that the RLC not but only to form RLC-RLC RLC and ELC for dephosphorylated and thiophosphorylated and to HMM in irradiation are at the of set of that irradiation of HMM and has effect the ATPase activity of This is also for myosin Sellers J.R. C.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). For shown are the is and is other where only one is it is with actin to the RLC or ELC not in are from the and the of the The RLC can as two with the labeled RLC the The exact of the the the of exchange and the and We have evidence that irradiation to within this RLC In contrast to the results for the dephosphorylated the for the thiophosphorylated evidence for a RLC-RLC dimer after irradiation in the of actin Therefore, the results for the suggest that is a conformational between the dephosphorylated and thiophosphorylated states. The dephosphorylated positions in the from one head to the other head to for whereas this in the thiophosphorylated state. by RLC this general of was observed for the and and for the and was not observed with the ELC not these two mutants dephosphorylated and Q15C, also of an RLC-RLC dimer after irradiation in the of The two other mutants and a behavior to those A23C, and WT In the dephosphorylated was observed by not or RLC and for S59C in the of ELC was not observed not mutants and also and the observed for dephosphorylated S59C in the of actin was Therefore, in the of actin for these mutants not a between the dephosphorylated and thiophosphorylated states. the conformational differences between dephosphorylated and thiophosphorylated that were detected for Q15C, A23C, and WT (Cys-108) be detected for a catalytic To this was exchanged a truncated that both catalytic domains R. and J. R. in but the full RLC and ELC binding and both the RLC and found for the irradiation of the truncated HMM generated RLC-RLC dimers in the dephosphorylated whereas RLC-RLC dimers were detected in the thiophosphorylated with more with ELC not evidence for the truncated the with to the of photocross-linking to the phosphorylation state, suggesting that the conformational change detected not the catalytic domains. the results from this We these as residues an with the chicken skeletal RLC sequence and structure (21Rayment I. Rypniewski W.R. Schmidt-Base K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 1-58Google Scholar). also as residues in with the scallop regulatory domain with the binding were because binding of the RLC for the heavy of the mutants were found to exchange to and to be well regulated by thiophosphorylation suggesting that the structural of the mutant were to the of RLC-RLC RLC on in of in of catalytic that an RLC-RLC was whereas a was detected or a in was not in a that an RLC-RLC was whereas a was detected or a in was not The labeled RLC mutants to into two classes. In the of HMMs exchanged with the labeled mutants Q15C, and WT (Cys-108) formed RLC-RLC dimers after irradiation but only in the dephosphorylated state. was not (Q15C and or The of the from the cysteine to the is about 8.9 we that the dephosphorylated for these regions of the RLC to approach the RLC of the other head to within this The of the RLC domain is about Our of the of in the thiophosphorylated is that these regions of the RLC are from one We not the that thiophosphorylation changes the of selectively because of the behavior of the G. Biochemistry. 1994; PubMed Scopus Google Scholar) and because the general behavior was observed for mutants in this Our results are not with the that the phosphorylated has about the head-tail This is because may with this were we have to in the thiophosphorylated for mutant positions mutants that show in It is that we observed phosphorylation-dependent at and in the of This that was not to a because the is not at and also the of Sellers J.R. C.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). and not the RLC-RLC Our photocross-linking was by thiophosphorylation and actin The Q15C mutant was also exchanged into a truncated nonmuscle HMM lacking both catalytic domains. found for the HMM, RLC-RLC was observed in the dephosphorylated suggesting that the catalytic domain is not for the to the between the RLC domains. This is with the of effect of Our also on a an in of the RLC domain phosphorylation J. S. Sweeney H.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, the catalytic domains are not required for phosphorylation-dependent changes in between the two RLC domains. In the second of S59C and was not observed in the dephosphorylated or thiophosphorylated we that these regions of the RLC are from the of the RLC that to approach as by the of mutants. and are in the and at the of the these two residues are on of the RLC from the residues in this little information been about the positions of the NH2-terminal region of the RLC, including and Ala-23, the thiophosphorylated residue because the residues from the skeletal myosin S1 (21Rayment I. Rypniewski W.R. Schmidt-Base K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 1-58Google Scholar) and the scallop regulatory domain (22Xie X. Harrison D.H. Schlichting I. Sweet R.M. Kalabokis V.N. Szent-Gyorgyi A.G. Cohen C. Science. 1994; 368: 306-312Google Scholar) were found to be The region of the smooth muscle RLC is residues in results with the Q15C and it is that at one of the interactions of this region is in approach to the other the positions of NH2-terminal and is at the of the within the COOH-terminal domain of the RLC as by the between the and (21Rayment I. Rypniewski W.R. Schmidt-Base K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 1-58Google Scholar). It is that be to A23C, and Cys-108 are on the of the RLC and that both have the behavior of the head-tail junction with two scallop myosin regulatory domains (22Xie X. Harrison D.H. Schlichting I. Sweet R.M. Kalabokis V.N. Szent-Gyorgyi A.G. Cohen C. Science. 1994; 368: 306-312Google Scholar) to a of the scallop tail, interactions between the two RLC G. P. J. Mol. Biol. 1996; PubMed Scopus Google Scholar) in a This to S59C to residues in the COOH-terminal domain of the RLC of the other In S59C RLC-RLC suggesting that at in this the is not within 8.9 The scallop also not the we from in general results are not in with the scallop The general effect of actin irradiation for the mutants was to RLC-RLC in the thiophosphorylated states. For the second of actin effect or for in the dephosphorylated (S59C). These were with a of actin HMM We have not the observed in the is or by the of it is that it is as in HMM of the results in HMM thiophosphorylation for RLC domain is in the complex, the head-head is This of HMM heads to actin be these the skeletal structure as a (21Rayment I. Rypniewski W.R. Schmidt-Base K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 1-58Google Scholar), two heads attached to in the coiled-coil tail to for of the RLC domains. Our results suggest that for smooth muscle HMM, the of the heads to actin from this in the region of the RLC, for proximity of the two In the S1 (21Rayment I. Rypniewski W.R. Schmidt-Base K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 1-58Google Scholar) is with between the catalytic domains observed in the smooth H. T. G. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). are required to the for It has been that full regulation of SMM interaction of the heads with the of the tail (20Trybus K.M. Naroditskaya V. Sweeney H.L. J. Biol. Chem. 1998; 273: 18423-18428Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) in the dephosphorylated state. However, results from mutants of the of the suggest that a acid sequence at the junction may not be critical for full regulation M. M. Mabuchi K. Kambara T. Ikebe R. Biochemistry. 1998; PubMed Scopus Google Scholar). It is to at for the mutants we not chain suggesting that the mutant residues not approach the heavy chain within 8.9 of smooth muscle myosin also not show evidence for interaction of the RLC with the tail region J. 1997; Full Text PDF PubMed Scopus Google Scholar). It is to results into the of studies of the of phosphorylation on the structure of regulated HMMs. It is to results with myosin because of to the and forms that are not to the HMM To phosphorylation of smooth muscle HMM has been to the H. Stafford II, W.F. J.C. J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar), a that appears to have the heads from the tails contrast to heads the H. Stafford II, W.F. J.C. J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar), proteolytic of the head-tail junction M. Hartshorne D.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the of the active region and the RLC J. S. Sweeney H.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). that the of between the two heads was by thiophosphorylation of smooth muscle myosin at J. 1997; Full Text PDF PubMed Scopus Google Scholar). these studies show an dephosphorylated head-tail junction, with and a more structure the phosphorylated state. with scallop HMM show by binding R. J. Cell Scholar). However, from these low studies it is not possible to interactions by phosphorylation. Our information through the use of a chemical that information about within the RLC The labeled mutants a to dephosphorylated smooth HMM that a of the two RLC and a of the RLC phosphorylation. to be at the between the two within 8.9 Our not between a interaction and one that is but within of the photocross-linking We found that are unable to in HMM, is behavior for probes that not have to the between the We are the of the photocross-linking to the of and to further examine the of the RLC in are to with other We for for with of the light chain and and for the We G. for the of the of the head-tail