FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2044107689> ?p ?o ?g. }

• W2044107689 endingPage "14475" @default.
• W2044107689 startingPage "14469" @default.
• W2044107689 abstract "The N terminus of skeletal myosin light chain 1 and the cardiomyopathy loop of human cardiac myosin have been shown previously to bind to actin in the presence and absence of tropomyosin (Patchell, V. B., Gallon, C. E., Hodgkin, M. A., Fattoum, A., Perry, S. V., and Levine, B. A. (2002) Eur. J. Biochem. 269, 5088–5100). We have extended this work and have shown that segments corresponding to other regions of human cardiac β-myosin, presumed to be sites of interaction with F-actin (residues 554–584, 622–646, and 633–660), likewise bind independently to actin under similar conditions. The binding to F-actin of a peptide spanning the minimal inhibitory segment of human cardiac troponin I (residues 134–147) resulted in the dissociation from F-actin of all the myosin peptides bound to it either individually or in combination. Troponin C neutralized the effect of the inhibitory peptide on the binding of the myosin peptides to F-actin. We conclude that the binding of the inhibitory region of troponin I to actin, which occurs during relaxation in muscle when the calcium concentration is low, imposes conformational changes that are propagated to different locations on the surface of actin. We suggest that the role of tropomyosin is to facilitate the transmission of structural changes along the F-actin filament so that the monomers within a structural unit are able to interact with myosin. The N terminus of skeletal myosin light chain 1 and the cardiomyopathy loop of human cardiac myosin have been shown previously to bind to actin in the presence and absence of tropomyosin (Patchell, V. B., Gallon, C. E., Hodgkin, M. A., Fattoum, A., Perry, S. V., and Levine, B. A. (2002) Eur. J. Biochem. 269, 5088–5100). We have extended this work and have shown that segments corresponding to other regions of human cardiac β-myosin, presumed to be sites of interaction with F-actin (residues 554–584, 622–646, and 633–660), likewise bind independently to actin under similar conditions. The binding to F-actin of a peptide spanning the minimal inhibitory segment of human cardiac troponin I (residues 134–147) resulted in the dissociation from F-actin of all the myosin peptides bound to it either individually or in combination. Troponin C neutralized the effect of the inhibitory peptide on the binding of the myosin peptides to F-actin. We conclude that the binding of the inhibitory region of troponin I to actin, which occurs during relaxation in muscle when the calcium concentration is low, imposes conformational changes that are propagated to different locations on the surface of actin. We suggest that the role of tropomyosin is to facilitate the transmission of structural changes along the F-actin filament so that the monomers within a structural unit are able to interact with myosin. One of the outstanding problems of muscle is to define in protein structural terms how actin interacts with myosin. This interaction enables the conversion of myosin from an enzyme that in the resting muscle hydrolyzes its substrate, MgATP, at a very low rate to one with the high rate associated with contraction. These features are intrinsic to the regulatory process. Crystals of actomyosin or the myosin head (S1) complexed with actin are not yet available to permit the determination of the high resolution structure of the myosin motor domain bound to actin. Nevertheless, modeling of the interaction using the known structures of actin and myosin S1, mutation studies, and a variety of experimental approaches have highlighted a number of regions on the myosin motor domain that may be involved in the interaction (1Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Science. 1993; 261: 58-65Crossref PubMed Scopus (1449) Google Scholar, 2Milligan R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 21-26Crossref PubMed Scopus (157) Google Scholar, 3Sasaki N. Asukagawa H. Yasuda R. Hiratsuka T. Sutoh K. J. Biol. Chem. 1999; 274: 37840-37844Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 4Volkmann N. Hanein D. Ouyang G. Trybus K.M. DeRosier D.J. Lowey S. Nat. Struct. Biol. 2000; 7: 1147-1155Crossref PubMed Scopus (135) Google Scholar, 5Joel P.B. Sweeney H.L. Trybus K.M. Biochemistry. 2003; 42: 9160-9166Crossref PubMed Scopus (32) Google Scholar, 6Joel P.B. Trybus K.M. Sweeney H.L. J. Biol. Chem. 2001; 276: 2998-3003Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 7Holmes K.C. Angert I. Kull F.J. Jahn W. Schroder R.R. Nature. 2003; 425: 423-427Crossref PubMed Scopus (311) Google Scholar, 8Holmes K.C. Schroder R.R. Sweeney H.L. Houdesse A. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2004; 359: 1819-1828Crossref PubMed Scopus (123) Google Scholar, 9Houdusse A. Szent-Gyorgyi A.G. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11238-11243Crossref PubMed Scopus (284) Google Scholar, 10Yengo C.M. Chrin L. Rovner A.S. Berger C.L. Biochemistry. 1999; 38: 14515-14523Crossref PubMed Scopus (33) Google Scholar, 11Onishi H. Morales M. Katoh K. Fujiwara K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11965-11969Crossref PubMed Scopus (17) Google Scholar, 12Mornet D. Pantel P. Audemard E. Kassab R. Biochem. Biophys. Res. Commun. 1979; 89: 925-932Crossref PubMed Scopus (189) Google Scholar, 13Uyeda T.Q.P. Ruppel K.M. Spudich J.A. Nature. 1994; 368: 567-569Crossref PubMed Scopus (188) Google Scholar, 14Knetsch M.L.W. Uyeda T.Q.P. Manstein D.J. J. Biol. Chem. 1999; 274: 20133-20138Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 15Van Dijk J. Furch M. Lafont C. Manstein D.J. Chaussepied P. Biochemistry. 1999; 38: 15078-15085Crossref PubMed Scopus (28) Google Scholar). It was concluded from the original structural studies on chicken skeletal myosin S1 that the interface with actin is likely to involve at least three exposed segments common to members of the myosin family (1Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Science. 1993; 261: 58-65Crossref PubMed Scopus (1449) Google Scholar, 2Milligan R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 21-26Crossref PubMed Scopus (157) Google Scholar). Implicated in making contact with a single actin subunit were the regions comprising chicken skeletal myosin residues 403–416 (the so-called “cardiomyopathy” loop), the helix-turn-helix region, residues 529–558, and residues 626–647 (loop 2, at the protease-sensitive junction between the 50- and 20-kDa domains). Another surface loop, residues 567–578, has been modeled as interacting with an adjacent actin monomer (1Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Science. 1993; 261: 58-65Crossref PubMed Scopus (1449) Google Scholar, 2Milligan R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 21-26Crossref PubMed Scopus (157) Google Scholar, 4Volkmann N. Hanein D. Ouyang G. Trybus K.M. DeRosier D.J. Lowey S. Nat. Struct. Biol. 2000; 7: 1147-1155Crossref PubMed Scopus (135) Google Scholar). The short cardiomyopathy loop of myosin is resolved as a well defined surface protrusion that is modeled as docking onto actin upon strong binding of myosin (4Volkmann N. Hanein D. Ouyang G. Trybus K.M. DeRosier D.J. Lowey S. Nat. Struct. Biol. 2000; 7: 1147-1155Crossref PubMed Scopus (135) Google Scholar, 7Holmes K.C. Angert I. Kull F.J. Jahn W. Schroder R.R. Nature. 2003; 425: 423-427Crossref PubMed Scopus (311) Google Scholar, 8Holmes K.C. Schroder R.R. Sweeney H.L. Houdesse A. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2004; 359: 1819-1828Crossref PubMed Scopus (123) Google Scholar). This loop region is clearly significant for actomyosin interaction because residue deletion results in the loss of the strong binding of actin to myosin (3Sasaki N. Asukagawa H. Yasuda R. Hiratsuka T. Sutoh K. J. Biol. Chem. 1999; 274: 37840-37844Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The residue change Arg → Gln in this loop of the β-chain of human cardiac myosin is associated with familial hypertrophic cardiomyopathy (16Geisterfer-Lowrance A.A.T. Kass S. Tanigawa G. Vosberg H-P. McKenna W. Seidman C.E. Seidman J.G. Cell. 1990; 62: 999-1006Abstract Full Text PDF PubMed Scopus (1039) Google Scholar, 17Cuda G. Fananapazir L. Zhu W.S. Sellers J.R. Epstein N.D. J. Clin. Investig. 1993; 91: 2861-2865Crossref PubMed Scopus (202) Google Scholar) and has been reported to result in altered kinetic properties of the actin-activated myosin ATPase (18Cuda G. Fananapazir L. Epstein N.D. Sellers J.R. J. Muscle Res. Cell Motil. 1997; 18: 275-283Crossref PubMed Scopus (113) Google Scholar, 19Sweeney H.L. Rosenfeld S.S. Brown F. Faust L. Smith J. Xing J. Stein L.A. Sellers J.R. J. Biol. Chem. 1998; 273: 6262-6270Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). We here use a peptide mapping approach to probe the sequence determinants of the interaction of surface loops of myosin with F-actin. Peptides comprising the cardiomyopathy loop of human cardiac β-myosin (hcβM 1The abbreviations used are: hcβM, human cardiac β-myosin; hcTnI, human cardiac troponin-I; rcTnI, rabbit cardiac troponin-I; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; IAEDANS, ((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid.1The abbreviations used are: hcβM, human cardiac β-myosin; hcTnI, human cardiac troponin-I; rcTnI, rabbit cardiac troponin-I; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; IAEDANS, ((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid.-(398–414)) and the N terminus of striated muscle myosin light chain 1 interact with F-actin (20Timson D.J. Trayer I.P. FEBS Lett. 1997; 400: 31-36Crossref PubMed Scopus (24) Google Scholar, 21Morano I. Haase H. FEBS Lett. 1997; 408: 71-74Crossref PubMed Scopus (36) Google Scholar, 22Patchell V.B. Gallon C.E. Hodgkin M.A. Fattoum A. Perry S.V. Levine B.A. Eur. J. Biochem. 2002; 269: 5088-5100Crossref PubMed Scopus (14) Google Scholar, 23Bartegi A. Roustan C. Chavanieu A. Kassab R. Fattoum A. Eur. J. Biochem. 1997; 250: 484-491Crossref PubMed Scopus (9) Google Scholar) and are able to modulate the actin-activated MgATPase of myosin (23Bartegi A. Roustan C. Chavanieu A. Kassab R. Fattoum A. Eur. J. Biochem. 1997; 250: 484-491Crossref PubMed Scopus (9) Google Scholar, 24Rarick H.M. Opgenorth T.J. vonGeldern T.W. WuWong J.S.R. Solaro R.J. J. Biol. Chem. 1996; 271: 27039-27043Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The affinity of these peptides for F-actin was observed to increase slightly in the presence of tropomyosin, whereas their interaction was prevented by the binding of the inhibitory region of troponin I to actin (22Patchell V.B. Gallon C.E. Hodgkin M.A. Fattoum A. Perry S.V. Levine B.A. Eur. J. Biochem. 2002; 269: 5088-5100Crossref PubMed Scopus (14) Google Scholar). We have extended these observations to peptides representing the other presumptive binding regions of myosin, and we have investigated their individual actin-binding properties. Our results indicate that three loops on the surface of the myosin molecule, human cardiac β-myosin residues 398–414, 554–584, and 623–660, interact with distinct sites on the actin molecule. These interactions provided the opportunity to explore the regulatory effects of tropomyosin and troponin-I. In the case of each myosin peptide, the nature of the residues making contact with F-actin was unaltered by tropomyosin. Interaction of the inhibitory region of human cardiac troponin-I with F-actin in the absence or presence of tropomyosin displaced all three myosin contacts. Some aspects of this work have been briefly reported in abstract form elsewhere (25Gallon C.E. Patchell V.B. Levine B.A. Perry S.V. J. Muscle Cell Motil. 2003; 24: 327Google Scholar). Peptides—The peptides of human cardiac β-myosin and human cardiac troponin-I (hcTnI) used in this study (Table I) were synthesized with acetylated N- and amidated C-terminal residues (Alta Bioscience, Birmingham University) and were purified as described previously (22Patchell V.B. Gallon C.E. Hodgkin M.A. Fattoum A. Perry S.V. Levine B.A. Eur. J. Biochem. 2002; 269: 5088-5100Crossref PubMed Scopus (14) Google Scholar). The composition and purity of all peptides were confirmed by mass spectral and NMR analyses. Amino acid analysis was used to determine the concentration of each of the stock peptide solutions used for the fluorescence and NMR studies.Table ISequences of the peptides of hcβM, human and rabbit cardiac troponin I (hcTnI, rcTnI) used in this studyPeptideSequencehcβM-(291-311)SNKKPELLDMLLITNNPYDYAhcβM-(365-388)KLKQREEQAEPDGTEEADKSAYLMhcβM-(398-414)GLCHPRVKVGNEYVTKGhcβM-(554-584)DNHLGKSANFQKPRNIKGKPEAHFSLIHYAGhcβM-(622-646)ANYAGADAPIEKGKGKAKKGSSFQThcβM-(633-660)KGKGKAKKGSSFQTVSALHRENLNKLMThcTnI-(128-153)TQKIFDLRGKFKRPTLRRVRISADAMhcTnI-(128-140)TQKIFDLRGKFKRhcTnI-(141-153)PTLRRVRISADAMrcTnI-(164-184)AKETLDLRAHLKQVKKEDTEK Open table in a new tab Muscle Proteins—Actin was isolated freeze-dried from rabbit skeletal muscle using the method of Spudich and Watt (26Spudich J.A. Watt S. J. Biol. Chem. 1971; 264: 4866-4871Abstract Full Text PDF Google Scholar). Measurement of the absorbance of G-actin at 290 nm was used to determine the concentration of the G-actin in the solution. The actin concentration was calculated using the empirical observation that absorbance at 290 nm of a 1 mg/ml solution is equal to 0.63 units (26Spudich J.A. Watt S. J. Biol. Chem. 1971; 264: 4866-4871Abstract Full Text PDF Google Scholar). F-actin was prepared in a solution containing 2 mm MgCl2 and 50 mm KCl as described previously (22Patchell V.B. Gallon C.E. Hodgkin M.A. Fattoum A. Perry S.V. Levine B.A. Eur. J. Biochem. 2002; 269: 5088-5100Crossref PubMed Scopus (14) Google Scholar) and dialyzed overnight against several changes of 5 mm sodium phosphate buffer, pH 7.0, in H2O2 for fluorescence measurements or H2O for the NMR titration studies. The F-actin-tropomyosin complex was made by adding G-actin to a stock solution of 1 mg/ml rabbit skeletal tropomyosin in 50 mm Tris-HCl, pH 7.6, 100 mm KCl, to give a final concentration of 2.5 mg/ml actin, 0.5 mg/ml tropomyosin. The complex was dialyzed into several changes of 5 mm phosphate buffer, pH 7.0, in 2H2O. Complex formation and the absence of free protein was confirmed by comparison with the electrophoretic patterns of the free proteins obtained upon electrophoresis on 10% nondenaturing polyacrylamide gels run in 10% (v/v) glycerol, 25 mm Tris, 80 mm glycine, pH 8.3. G-actin labeled at Cys-374 with IAEDANS was prepared according to the method of Miki et al. (27Miki M. dos Remedios C.G. Barden J.A. Eur. J. Biochem. 1987; 168: 339-345Crossref PubMed Scopus (57) Google Scholar) as reported previously (22Patchell V.B. Gallon C.E. Hodgkin M.A. Fattoum A. Perry S.V. Levine B.A. Eur. J. Biochem. 2002; 269: 5088-5100Crossref PubMed Scopus (14) Google Scholar). The extent of labeling was normally 0.8–0.9 mol/mol G-actin. This preparation of labeled G-actin was polymerized using 50 mm KCl, 2 mm MgCl2, and aliquots were stored frozen for subsequent use. Fluorescence Measurements—All fluorescence emission spectra were obtained using a PerkinElmer Life Sciences LS50B fluorescence spectrometer. Emission fluorescence intensity values (IAEDANS excitation at 340 nm) were corrected for solvent emission fluorescence and any dilution effects (<5%) resulting from the titration carried out by the sequential addition of small (1–5 μl) volumes of the peptide solution. The fractional change in fluorescence observed was assumed to be directly proportional to the fraction of the complex formed. The dissociation constant was derived using a nonlinear regression procedure fitting the fluorescence data obtained in separate titrations to a 1:1 binding curve. NMR Studies—All spectra were obtained using a Bruker 500-MHz DRX spectrometer equipped with a cryoprobe. The residue-specific assignment of peptide resonances was carried out using standard protocols following the acquisition of total correlation spectroscopy and NOESY spectra using samples in 90% H2O solution. Titration of the myosin peptides (concentration range used 40–300 μm) with F-actin was typically carried out by sequential addition of aliquots of F-actin (10 mg/ml) or F-actin-tropomyosin (5 mg/ml F-actin) at T = 293 K in 2H2O solution, pH 7.4, buffered with 25 mm Tris HCl. The molar proportions of the components of each mixture were obtained from the respective concentrations in solution. The broad signals of the spectrum of F-actin obtained upon these additions contributed relatively little to the spectra of the peptides in the presence of actin. Confirmation of the extent of relaxation resulting from interaction with F-actin was obtained by use of two-pulse spin-echo spectra (1024 transients) using a (90–180-t) sequence with a delay time, t = 60 ms, and an overall delay of 3 s to enable complete magnetization recovery. In these experiments the coupling constant and relaxation time of individual resonances modulate the signal amplitudes that therefore provide sensitive indicators of the extent of complex formation. The spectral perturbations were also visualized by difference spectra taken at each stage of the titration. The identification of contact residues on a small peptide interacting with a much larger protein can be undertaken by the use of NMR spectroscopy to detect the broadening of the signals from residues of the peptide at the molecular interface. This broadening is a consequence of the much faster relaxation rate in the bound state and the dynamic process of exchange between free and bound states of the target peptide (28Lian L-Y. Roberts G.C.K. Roberts G.C.K. NMR of Macromolecules: A Practical Approach. Oxford University Press, New York1993: 153-182Google Scholar, 29Zuiderweg E.R.P. Biochemistry. 2002; 41: 1-7Crossref PubMed Scopus (466) Google Scholar, 30Jardetzky O. Roberts G.C.K. NMR in Molecular Biology. Academic Press, New York1981Google Scholar). The interface with F-actin can be characterized by the progressive perturbation of specific peptide resonances during titration that directly reflects the population of the free and bound states of each peptide. The use of a cryogenic probe for such experiments permits titrations to be carried out using concentrations in the low micromolar range thereby reducing the probability of nonspecific interaction. Titrations monitored by both NMR and fluorescence have been used to confirm that binding is saturable and of defined stoichiometry. The directness of this experimental approach has allowed us to investigate the F-actin and actin-tropomyosin binding characteristics of different regions of the human β-cardiac myosin head and the modulation of these actin associations by the inhibitory region of human cardiac troponin-I. Multiple Contacts of Residues 398–414 of Human Cardiac β-Myosin with F-actin Involve a Preference for a Looped Conformation—In view of the importance of the cardiomyopathy loop region in the activation of the myosin ATPase (e.g. Refs. 3Sasaki N. Asukagawa H. Yasuda R. Hiratsuka T. Sutoh K. J. Biol. Chem. 1999; 274: 37840-37844Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar and 7Holmes K.C. Angert I. Kull F.J. Jahn W. Schroder R.R. Nature. 2003; 425: 423-427Crossref PubMed Scopus (311) Google Scholar) and the ability of the cardiac myosin peptide hcβM-(398–414) (Table I) to competitively inhibit actin-activated myosin ATPase (23Bartegi A. Roustan C. Chavanieu A. Kassab R. Fattoum A. Eur. J. Biochem. 1997; 250: 484-491Crossref PubMed Scopus (9) Google Scholar), we have defined the nature of this interaction in detail by carrying out titrations with F-actin by using a range of concentrations of the peptide (40–300 μm). As shown in Fig. 1, the presence of sub-stoichiometric concentrations of F-actin resulted in extensive signal broadening with spectral line widths dominated by the bound ligand cross-relaxation that results from the substantial increase in correlation time of the peptide bound to F-actin. The progressive line broadening observed with increasing concentration of F-actin (Fig. 1) reports on the relative population of free and bound states of the peptide, and the interaction interface can be robustly defined by inspection of the progressively broadened resonances. Notably affected at low ratios of complexed to free peptide are the side chain signals of Gly-407 and Asn-408 with the resonances deriving from His-401, Arg-403, Glu-409, Tyr-410, Val-411, and Thr-412 increasingly broadened in the presence of higher concentrations of F-actin (Fig. 1). The observation of line width perturbation for all of these uniquely resolved peptide signals in the presence or absence of tropomyosin indicates that hcβM-(398–414) lies close to the actin surface along most of its length. Of significance with regard to the extensive nature of the association between hcβM-(398–414) and F-actin was the NOESY-based evidence that the sequence Val-406 to Asn-408 in the middle of the peptide was characterized by a predisposition to adopt a β-turn conformation. Clearly resolved in the amide region of the proton NOESY spectrum is the (i → i + 2) NOE cross-peak that identifies the time-average proximity of the side chain methyl group(s) of Val-406 and the backbone –NH of Asn-408 (Fig. 2). The relatively strong intensity of this (i → i + 2) cross-peak compared with the variety of inter-residue (i → i + 1) NOE detected suggests the presence of a significant population of the turn conformation of the central region of the peptide. This conclusion is supported by the further observation of the nonequivalence of the resonances of the two Cα protons of Gly-407 (Fig. 2) that is indicative of a preferred rotamer population (30Jardetzky O. Roberts G.C.K. NMR in Molecular Biology. Academic Press, New York1981Google Scholar). Because the sequences flanking the central turn region of hcβM-(398–414) contain charged residues, we proceeded to investigate the sensitivity of the interaction of F-actin with the peptide to ionic strength. These spectral studies showed that the addition of salt did not significantly alter the structured predisposition of the central region of the peptide, and we observed that the interaction is salt concentration-dependent, with the actin-induced perturbation of the different residues along the peptide chain virtually undetectable in 50 mm KCl (Fig. 3). Earlier binding studies using peptides incorporating the site mutations R403Q and E407S have shown that actin affinity remained unaffected by these single charge changes with randomization of the peptide sequence resulting in the loss of actin binding (23Bartegi A. Roustan C. Chavanieu A. Kassab R. Fattoum A. Eur. J. Biochem. 1997; 250: 484-491Crossref PubMed Scopus (9) Google Scholar). Although our results indicate multiple contacts with F-actin along the sequence of hcβM-(398–414), they do not preclude the possibility that the N-terminal and C-terminal regions of this peptide bind to actin with differing affinity. Complex formation by hcβM-(398–414), however, appears to be mediated by electrostatic contributions, a characteristic previously only associated with the interaction of loop 2 of myosin with F-actin (5Joel P.B. Sweeney H.L. Trybus K.M. Biochemistry. 2003; 42: 9160-9166Crossref PubMed Scopus (32) Google Scholar, 6Joel P.B. Trybus K.M. Sweeney H.L. J. Biol. Chem. 2001; 276: 2998-3003Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 12Mornet D. Pantel P. Audemard E. Kassab R. Biochem. Biophys. Res. Commun. 1979; 89: 925-932Crossref PubMed Scopus (189) Google Scholar, 13Uyeda T.Q.P. Ruppel K.M. Spudich J.A. Nature. 1994; 368: 567-569Crossref PubMed Scopus (188) Google Scholar, 14Knetsch M.L.W. Uyeda T.Q.P. Manstein D.J. J. Biol. Chem. 1999; 274: 20133-20138Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 15Van Dijk J. Furch M. Lafont C. Manstein D.J. Chaussepied P. Biochemistry. 1999; 38: 15078-15085Crossref PubMed Scopus (28) Google Scholar). Localization of the Residues of Loop 2 of Human Cardiac β-Myosin That Interact with F-actin—Two overlapping segments of the loop 2 region of human cardiac β-myosin, peptide 600A, residues 622–646, and peptide 600B, residues 633–660 (Table I), were studied to compare the nature of their interaction with F-actin. Perturbation of residues 633–646 in the overlapping region was observed with both peptides in the presence of F-actin. Although neither peptide was found to interact with tropomyosin in control titrations, comparable spectral broadening in the case of each peptide occurred at lower F-actin:peptide molar ratios when tropomyosin was present. These observations indicated that tropomyosin had increased the actin-binding affinity of each peptide but had not altered the nature of the residues involved in the association with F-actin. In the case of hcβM-(622–646), a very restricted subset of peptide residues was observed to interact with F-actin (Fig. 4). Marked spectral perturbation occurred for the extreme C-terminal residues Gly-641, Phe-644, Gln-645, and Thr-646. Resonances of residues at the N-terminal of hcβM-(622–646), Tyr-624, Ala-629, Ile-631, and Glu-632 remained virtually unaffected and even retained fine structure (Fig. 4B). These observations indicated that the N-terminal region of the bound peptide extended away from the actin surface and retained segmental mobility independent of F-actin. Inspection of the lysine ϵCH2 side chain resonance that is derived from the cluster of five basic residues adjacent to the C-terminal of hcβM-(622–646) showed that this composite signal was only slightly altered upon association with F-actin (Fig. 4B). The comparably small perturbation detected for Ala-638 and the clear demarcation observed between the actin-bound C-terminal residues and those at the mobile N terminus of hcβM-(622–646) suggested that the actin-induced spectral alteration in the composite lysine resonance could have resulted from the involvement in complex formation of Lys-639 and/or Lys-640 at the C terminus of the basic cluster. The interaction of hcβM-(622–646) with F-actin was, however, relatively stable to increasing ionic strength with complex formation virtually abolished only in the presence of 0.2 m KCl (Fig. 4C). Notably, acetylation of the lysine residues of hcβM-(622–646) resulted in the complete loss of actin binding with complex formation undetectable even when F-actin was present in solution at a 2.5 m excess (Fig. 4D). The actin affinity of hcβM-(622–646) was determined by monitoring the change in the fluorescence emission of IAEDANS-labeled F-actin (Fig. 5A) under solution conditions corresponding to the NMR experiment shown (Fig. 4B). Saturation of the quenching of the emission of the IAEDANS label attached to actin Cys-374 was observed with an apparent dissociation constant of 1 ± 0.5 μm derived for 1:1 complex formation. Taken overall, the observations are indicative of a specific mode of docking involving the C-terminal residues of hcβM-(622–646). These residues occur in the central region of hcβM-(633–660) whose interaction with F-actin showed that the composite lysine side chain signal was relatively unperturbed by complex formation (Fig. 5B), again suggestive of a fluctuating interaction with the surface of actin by a restricted subset of the cluster of basic residues at the N-terminal of loop 2 of human cardiac β-myosin. The association of hcβM-(633–660) with F-actin involved residues Phe-644, Val-647, His-651, Arg-652, Met-659, and Thr-660 downstream of the basic N terminus (Fig. 5B). Specificity of Interaction of the Myosin Loop Peptides with F-actin Is Demonstrated by the Failure of Other Surface-exposed Regions of Myosin S1 to Associate with Actin—Inspection of the published structures of the myosin head shows two other regions that jut away from the surface of the molecule in a location close to the cardiomyopathy loop and loop 2 protrusions. The corresponding sequences of human cardiac β-myosin, hcβM-(291–311) and hcβM-(365–388) (Table I), were therefore synthesized for study of their potential actin-binding properties. No detectable spectral changes resulted upon titration of either peptide with F-actin or with F-actin-tropomyosin (Fig. 6). Resonances of hcβM-(291–311) (e.g. Lys-293, Lys-294, Thr-304, Tyr-308, Tyr-310, and Ala-311) retained fine structures even when present at a concentration equivalent to an equimolar ratio of peptide:F-actin (Fig. 6, A and B). Similarly, no detectable actin-induced spectral perturbation was observed for signals of residues along the sequence of hcβM-(365–388) (Lys-365, Lys-367, Leu-366, Leu-387, Thr-368, Arg-369, and Tyr-386; Fig. 6, C and D). These experiments using hcβM-(291–311) and hcβM-(365–388) indicated the complete absence of any interactions with the surface of actin and provided robust evidence for the specificity of complex formation with F-actin observed with the cardiomyopathy and the loop 2 regions that are also located on the same face of the myosin head. Concurrent Interaction of Three Different Myosin Loop Peptides with F-actin Indicates That They Possess Distinct Binding Sites—In order to study actin-binding site selectivity, equimolar mixtures of different myosin peptides were titrated with actin and actin-tropomyosin. As well as the human cardiac β-myosin cardiomyopathy loop and loop 2 peptides, we made use of a peptide comprising human cardiac β-myosin residues 554–584 also located on the same surface of the myosin head and modeled to be an actin-binding region (1Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Science. 1993; 261: 58-65Crossref PubMed Scopus (1449) Google Scholar, 2Milligan R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 21-26Crossref PubMed Scopus (157) Google Sc" @default.
• W2044107689 created "2016-06-24" @default.
• W2044107689 creator A5001018193 @default.
• W2044107689 creator A5023551676 @default.
• W2044107689 creator A5049849218 @default.
• W2044107689 creator A5063307072 @default.
• W2044107689 creator A5074806174 @default.
• W2044107689 creator A5090672193 @default.
• W2044107689 date "2005-04-01" @default.
• W2044107689 modified "2023-09-30" @default.
• W2044107689 title "The Regulatory Effects of Tropomyosin and Troponin-I on the Interaction of Myosin Loop Regions with F-actin" @default.
• W2044107689 cites W104354955 @default.
• W2044107689 cites W1534892601 @default.
• W2044107689 cites W1538692246 @default.
• W2044107689 cites W1923110202 @default.
• W2044107689 cites W1974842879 @default.
• W2044107689 cites W1982579049 @default.
• W2044107689 cites W1991181780 @default.
• W2044107689 cites W1991296405 @default.
• W2044107689 cites W1999251970 @default.
• W2044107689 cites W2006406812 @default.
• W2044107689 cites W2010337484 @default.
• W2044107689 cites W2010436557 @default.
• W2044107689 cites W2018531588 @default.
• W2044107689 cites W2031294645 @default.
• W2044107689 cites W2031862654 @default.
• W2044107689 cites W2040301650 @default.
• W2044107689 cites W2043664506 @default.
• W2044107689 cites W2044629144 @default.
• W2044107689 cites W2052806957 @default.
• W2044107689 cites W2053430231 @default.
• W2044107689 cites W2058814066 @default.
• W2044107689 cites W2059268325 @default.
• W2044107689 cites W2061294816 @default.
• W2044107689 cites W2062323111 @default.
• W2044107689 cites W2067425020 @default.
• W2044107689 cites W2067750636 @default.
• W2044107689 cites W2080194310 @default.
• W2044107689 cites W2091438840 @default.
• W2044107689 cites W2094251607 @default.
• W2044107689 cites W2107917810 @default.
• W2044107689 cites W2127063208 @default.
• W2044107689 cites W2151357467 @default.
• W2044107689 cites W2157566126 @default.
• W2044107689 cites W2159830386 @default.
• W2044107689 cites W383489959 @default.
• W2044107689 doi "https://doi.org/10.1074/jbc.m414202200" @default.
• W2044107689 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15695827" @default.
• W2044107689 hasPublicationYear "2005" @default.
• W2044107689 type Work @default.
• W2044107689 sameAs 2044107689 @default.
• W2044107689 citedByCount "14" @default.
• W2044107689 countsByYear W20441076892013 @default.
• W2044107689 countsByYear W20441076892015 @default.
• W2044107689 crossrefType "journal-article" @default.
• W2044107689 hasAuthorship W2044107689A5001018193 @default.
• W2044107689 hasAuthorship W2044107689A5023551676 @default.
• W2044107689 hasAuthorship W2044107689A5049849218 @default.
• W2044107689 hasAuthorship W2044107689A5063307072 @default.
• W2044107689 hasAuthorship W2044107689A5074806174 @default.
• W2044107689 hasAuthorship W2044107689A5090672193 @default.
• W2044107689 hasBestOaLocation W20441076891 @default.
• W2044107689 hasConcept C114614502 @default.
• W2044107689 hasConcept C12554922 @default.
• W2044107689 hasConcept C125705527 @default.
• W2044107689 hasConcept C126322002 @default.
• W2044107689 hasConcept C184670325 @default.
• W2044107689 hasConcept C185592680 @default.
• W2044107689 hasConcept C205875245 @default.
• W2044107689 hasConcept C33923547 @default.
• W2044107689 hasConcept C36036425 @default.
• W2044107689 hasConcept C500558357 @default.
• W2044107689 hasConcept C55493867 @default.
• W2044107689 hasConcept C6997183 @default.
• W2044107689 hasConcept C71924100 @default.
• W2044107689 hasConcept C86803240 @default.
• W2044107689 hasConcept C95444343 @default.
• W2044107689 hasConceptScore W2044107689C114614502 @default.
• W2044107689 hasConceptScore W2044107689C12554922 @default.
• W2044107689 hasConceptScore W2044107689C125705527 @default.
• W2044107689 hasConceptScore W2044107689C126322002 @default.
• W2044107689 hasConceptScore W2044107689C184670325 @default.
• W2044107689 hasConceptScore W2044107689C185592680 @default.
• W2044107689 hasConceptScore W2044107689C205875245 @default.
• W2044107689 hasConceptScore W2044107689C33923547 @default.
• W2044107689 hasConceptScore W2044107689C36036425 @default.
• W2044107689 hasConceptScore W2044107689C500558357 @default.
• W2044107689 hasConceptScore W2044107689C55493867 @default.
• W2044107689 hasConceptScore W2044107689C6997183 @default.
• W2044107689 hasConceptScore W2044107689C71924100 @default.
• W2044107689 hasConceptScore W2044107689C86803240 @default.
• W2044107689 hasConceptScore W2044107689C95444343 @default.
• W2044107689 hasIssue "15" @default.
• W2044107689 hasLocation W20441076891 @default.
• W2044107689 hasOpenAccess W2044107689 @default.
• W2044107689 hasPrimaryLocation W20441076891 @default.
• W2044107689 hasRelatedWork W160988685 @default.
• W2044107689 hasRelatedWork W162570409 @default.

• next