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W1990804119 abstract "Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp501 in the relay loop. Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp501 in the relay loop. 2′(3′)-O-(N-methylanthraniloyl)- adenosine 5′-(β,γ-imidotriphosphate) adenosine 5′-O-(3-thiotriphosphate) N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Interaction of nucleotides with vertebrate skeletal muscle myosin is accompanied by an enhancement in tryptophan fluorescence that has long been exploited to determine the mechanism of nucleotide binding and hydrolysis (1Bagshaw C.R. Eccleston J.F. Eckstein F. Goody R.S. Gutfreund H. Trentham D.R. Biochem. J. 1974; 141: 351-364Crossref PubMed Scopus (227) Google Scholar, 2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar, 3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar). Nonhydrolyzable nucleotides (e.g. ADP) cause a smaller enhancement than hydrolyzable ones (e.g. ATP), indicating that part of the fluorescence change observed with ATP may be associated with the hydrolysis step itself. The kinetics of binding saturate at high nucleotide concentrations, indicating that the process is at least two-step (1Bagshaw C.R. Eccleston J.F. Eckstein F. Goody R.S. Gutfreund H. Trentham D.R. Biochem. J. 1974; 141: 351-364Crossref PubMed Scopus (227) Google Scholar). Under some conditions, the fluorescence change associated with hydrolysis was resolved from a faster isomerization step (2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar). A more precise mechanism (Reaction FR1) for the fluorescence enhancement associated with hydrolysis has recently been proposed using a Dictyostelium discoideummyosin construct containing a single tryptophan residue at position 501 in the relay loop, some 3 nm from the active site (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar).We demonstrated that tryptophan at this position shows a small quench on nucleotide binding (10–20%) followed by a large enhancement (50–80%) associated with a major conformational change (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar), presumably equivalent to the open-closed transition identified by x-ray crystallography (6Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar). The latter step is freely reversible and slightly favors the open state (low fluorescence M†·ATP) in the presence of ATP under ambient conditions, but coupling to the hydrolysis step pulls the equilibrium over to the more stable M*ADP·Pi state with enhanced fluorescence (5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). Thus, under most conditions, the kinetics of Trp501 enhancement upon interaction with ATP provide a measure of the hydrolysis reaction, in line with earlier schemes (2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar), although relaxation methods reveal these processes to be distinct events (5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, 7Urbanke C. Wray J. Biochem. J. 2001; 358: 165-173Crossref PubMed Scopus (40) Google Scholar). The quench in Trp501 fluorescence that precedes the enhancement is related to the binding process, but details of this step are not easy to characterize because the signal is small and is obscured by the subsequent open-closed transition. In the present study, we have made constructs containing single tryptophan residues near the active site with the aim of probing the initial binding step(s) without any contribution from the conformational changes that are coupled to hydrolysis.The D. discoideum motor domain lacks tryptophan residues near the nucleotide site equivalent to positions Trp113 and Trp131 in vertebrate skeletal myosin. This finding has led to the suggestion that the enhancement seen upon ADP binding to vertebrate skeletal myosin may arise from specific perturbation of the tryptophan residues near the active site (6Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar, 8Kurzawa S. E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar). In skeletal muscle myosin, Trp131 lies in the vicinity of the adenine ring of the bound nucleotide, whereas the Trp113 side chain could be sensitive to nucleotide binding via interaction with the conservative Lys130 residue. We therefore have explored this idea by making the appropriate single tryptophan constructs in the D. discoideum motor domain. We previously showed that substitution of all of the naturally occurring tryptophan residues with phenylalanine in the D. discoideum motor domain had a fairly limited effect on the kinetic pathway (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar), and hence adding back a single tryptophan residue as a site-directed probe at locations throughout the molecule is a potentially viable approach.DISCUSSIONDespite the long history (1Bagshaw C.R. Eccleston J.F. Eckstein F. Goody R.S. Gutfreund H. Trentham D.R. Biochem. J. 1974; 141: 351-364Crossref PubMed Scopus (227) Google Scholar, 2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar, 3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar), the assignment of tryptophan fluorescence changes to specific steps in the vertebrate skeletal muscle myosin and actomyosin ATPase pathways has been controversial and difficult to resolve for a number of reasons. At 20 °C, the rapid isomerization steps are lost within the dead time of the stopped flow apparatus, and only the fluorescence change associated with hydrolysis is clearly resolved. At lower temperatures, additional steps were resolved, which suggested that there are at least two binding isomerizations that are distinct from the hydrolysis step (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar). In the presence of actin, there are further complications arising from the potential of tryptophans in actin to contribute to the signal change, perturbations of myosin tryptophans that are sensitive to actin binding (2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar, 25Yengo C.M. Chrin L. Rovner A.S. Berger C.L. Biochemistry. 1999; 38: 14515-14523Crossref PubMed Scopus (33) Google Scholar), and the technical difficulty of the effect of light scattering changes on the measured fluorescence signal (26Millar N.C. Geeves M.A. Biochem. J. 1988; 249: 735-743Crossref PubMed Scopus (37) Google Scholar). Use of D. discoideum myosin II constructs has the advantage that tryptophan residues can be added or removed by mutagenesis to probe specific regions of the myosin motor domain. Here we focus on the response of tryptophan residues located near the myosin nucleotide binding site. We show that a tryptophan residue at position 129 or 131 responds with similar kinetics (after allowing for species differences) as previously characterized for rabbit skeletal myosin (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar), although the fluorescence change is in the opposite direction. The cause of the fluorescence enhancement on nucleotide binding seen with vertebrate myosins has therefore not been unambiguously identified. There are several candidates for the source of the signal change in vertebrate myosins. First, the two tryptophans in the vicinity of the binding site (Trp113 and Trp131 in rabbit skeletal myosin), which are conserved only among skeletal and cardiac muscle isoforms, are likely to be sensitive to nucleotide binding (6Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar, 8Kurzawa S. E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar), although their local environments may be different from those in D. discoideum myosin to give a net enhancement rather than quench. Park and Burghardt (21Park S. Burghardt T.P. Biochemistry. 2000; 39: 11732-11741Crossref PubMed Scopus (31) Google Scholar) isolated Trp131 emission in skeletal muscle subfragment 1 by means of elimination of its fluorescence contribution using site-specific chemical modification. Their data show that this side chain indeed responds with a ∼20% enhancement upon binding of nucleotide, but they excluded Trp131 as the tryptophan sensitive to ATP hydrolysis on the basis that the signal was similar in different nucleotide states. Based on structural data, Trp113 also cannot be excluded as a potential candidate responsible for nucleotide binding-induced signal changes. The contribution of these residues to the signal change is also indicated by the fact that wild-type D. discoideum myosin II, which lacks Trp131 (and Trp113), does not respond with a significant change in fluorescence on nucleotide binding (8Kurzawa S. E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar, 9Kuhlman P.A. Bagshaw C.R. J. Muscle Res. Cell Motil. 1998; 19: 491-504Crossref PubMed Scopus (36) Google Scholar). The single tryptophan residues of the D. discoideum myosin mutants presented in this study (Trp113, Trp129, and Trp131) were insensitive to the events of the ATPase cycle other than nucleotide binding. However, the combined contributions of Trp113 and Trp131 would lead to a net quench rather than enhancement on nucleotide binding.The conservative tryptophan residue in the relay loop of the motor domain (Trp510 in skeletal, Trp512 in smooth muscle, and Trp501 in D. discoideummyosin), which is sensitive to the open-closed transition, also shows a fluorescence change upon nucleotide binding. Skeletal and smooth muscle myosin relay loop tryptophans show 38 and 30% enhancement upon ADP addition, respectively (21Park S. Burghardt T.P. Biochemistry. 2000; 39: 11732-11741Crossref PubMed Scopus (31) Google Scholar, 22Yengo C.M. Chrin L.R. Rovner A.S. Berger C.L. J. Biol. Chem. 2000; 275: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In the W501+ single tryptophanD. discoideum myosin mutant, a 15–20% quench can be detected on this step (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). It is unlikely that other tryptophans are involved (e.g. the conserved Trp432 in D. discoideum, Trp440 in skeletal myosin) because the W501F and W501Y mutants show almost no fluorescence change upon adding nucleotide (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar,20Batra R. Manstein D.J. Biol. Chem. 1999; 380: 1017-1023Crossref PubMed Scopus (49) Google Scholar). Our data therefore indicate that (i) both the tryptophan(s) near the binding site and the relay loop residue contribute to the fluorescence change upon nucleotide binding prior to the open-closed transition, and (ii) the direction and extent of the fluorescence change of the responsive tryptophans is largely determined by the nonconservative environment surrounding the tryptophan side chain. Thus, we conclude that the global conformational changes undertaken by these myosins are conserved (on the basis of similar rate profiles and functional properties), and even the local movement of particular tryptophan residues could be similar, but the environment experienced by the tryptophan side chains during such movements will vary because of nonconserved residues around them.The acrylamide quenching experiments indicate that a tryptophan residue at position 129 is solvent-exposed, as expected from the crystal structures (17Rayment I. Rypniewski W.R. Schmidt-Bäse K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 50-58Crossref PubMed Scopus (1859) Google Scholar, 18Gulick A.M. Bauer C.B. Thoden J.B. Rayment I. Biochemistry. 1997; 36: 11619-11628Crossref PubMed Scopus (178) Google Scholar). Nevertheless, time-resolved anisotropy measurements indicate that this side chain is not freely mobile, and interconversion between rotamer states occurs on a time scale slower than net rotation of the motor domain, as was also noted for Trp501 (16Málnási-Csizmadia A. Kovács M. Woolley R.J. Botchway S.W. Bagshaw C.R. J. Biol. Chem. 2001; 276: 19483-19490Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Nucleotide binding is accompanied by a protection of Trp129 from collisional quenching by agents in solution, but this residue is now subject to increased static quenching, possibly by direct association with the adenine moiety.Regardless of the specific origin of the fluorescence change, the large quench observed on nucleotide binding to W129+ allows a more precise analysis of the mechanism than has hitherto been possible with wild-type proteins. Furthermore, the signal from Trp129 is not sensitive to the open-closed transition, which removes some of the ambiguity in the analysis. This is particularly important because much of the earlier data exploited analogs such as AMP-PNP and ATPγS to mimic the nonhydrolyzed ATP state, but it is now known that these analogs induce the closed state to a limited extent (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, 7Urbanke C. Wray J. Biochem. J. 2001; 358: 165-173Crossref PubMed Scopus (40) Google Scholar) and hence have a contribution from the signal traditionally assigned to the M*·ADP·Pi state (M**·ADP·Pi in skeletal myosin). ADP has less or no tendency to induce the closed state (5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, 7Urbanke C. Wray J. Biochem. J. 2001; 358: 165-173Crossref PubMed Scopus (40) Google Scholar) and is therefore the simplest nucleotide with which to explore the initial binding steps.In accordance with this concept, the overall profiles of different nucleotides binding to the W129+ construct were similar in character to that observed by Trybus and Taylor (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar) for ADP binding to skeletal myosin at 5 °C (at higher temperatures, the isomerization steps with skeletal myosin are too fast to resolve). The profiles of skeletal andD. discoideum myosins differ in detail in the near loss of the slow phase at high [nucleotide], which indicates that the M†1·L and M†2·L states have nearly the same fluorescence yield in D. discoideum myosin (hence at saturating [nucleotide], step 2b is optically silent). Furthermore, the k obs of the fast phase appeared to extrapolate back to give a large intercept value (250–1000 s−1), whereas it was close to zero in the Trybus and Taylor (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar) data. This aspect is difficult to characterize with accuracy, However, it can be accounted for by an additional weak but competitive nonproductive binding mode of the nucleotide as in ReactionFR2. Here, M#·L might represent a nucleotide bound to the active site in a noncompetent orientation that must dissociate and reassociate (or rearrange while bound) before forming the tightly bound form M†2·L.The fluorescence transients from the double tryptophan construct, W129+/W501+, show a clear distinction between the nucleotide binding steps and the open-closed transition that is coupled to hydrolysis, because the signals have opposite signs (cf. skeletal myosin, where they both give enhancements). Furthermore, the effective hydrolysis rate is slowed to 8 s−1 at 20 °C, which may be useful for developing conformationally resolved single molecule ATPase assays because the transition occurs on the time scale appropriate for video acquisition (27Bagshaw C.R. Conibear P.B. Single Mol. 2000; 1: 269-275Crossref Scopus (4) Google Scholar).Although the aim of this work was to characterize the tryptophan residue(s) in the motor domain that respond to the nucleotide binding steps and to exploit the fluorescence signal to delineate the elementary steps of this process, it is of interest to relate these steps to those that occur in the presence of actin. Previous studies have estimated that the ATP-induced dissociation of acto-D. discoideum motor domain is limited by a first order step of 150–450 s−1 at 20 °C (8Kurzawa S. E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar, 9Kuhlman P.A. Bagshaw C.R. J. Muscle Res. Cell Motil. 1998; 19: 491-504Crossref PubMed Scopus (36) Google Scholar) and is thus comparable with the slower of the two isomerization steps revealed by Trp129 fluorescence. We confirm that the acto-W129+ construct dissociates with a maximum rate constant of 150 s−1 at saturating [ATP]. The conformational changes sensed by the tryptophan residues near the active site (Trp129 and Trp131) are therefore likely to occur before or are coincident with the ATP-induced actomyosin dissociation reaction. This is in agreement with thermodynamic arguments that require coupling with an effectively irreversible step (step 2b with ATP) in the binding mechanism to provide sufficient energy to drive the actomyosin dissociation reaction. We find no evidence of a slower tryptophan fluorescence change sensed by Trp129 after actin dissociation, but previous reports with skeletal myosin where this was observed may reflect a contribution from the open-closed transition (26Millar N.C. Geeves M.A. Biochem. J. 1988; 249: 735-743Crossref PubMed Scopus (37) Google Scholar). Single tryptophan-containing constructs will provide a valuable tool for further delineating communication pathways between the actin site, nucleotide site, and converter region of myosins. Interaction of nucleotides with vertebrate skeletal muscle myosin is accompanied by an enhancement in tryptophan fluorescence that has long been exploited to determine the mechanism of nucleotide binding and hydrolysis (1Bagshaw C.R. Eccleston J.F. Eckstein F. Goody R.S. Gutfreund H. Trentham D.R. Biochem. J. 1974; 141: 351-364Crossref PubMed Scopus (227) Google Scholar, 2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar, 3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar). Nonhydrolyzable nucleotides (e.g. ADP) cause a smaller enhancement than hydrolyzable ones (e.g. ATP), indicating that part of the fluorescence change observed with ATP may be associated with the hydrolysis step itself. The kinetics of binding saturate at high nucleotide concentrations, indicating that the process is at least two-step (1Bagshaw C.R. Eccleston J.F. Eckstein F. Goody R.S. Gutfreund H. Trentham D.R. Biochem. J. 1974; 141: 351-364Crossref PubMed Scopus (227) Google Scholar). Under some conditions, the fluorescence change associated with hydrolysis was resolved from a faster isomerization step (2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar). A more precise mechanism (Reaction FR1) for the fluorescence enhancement associated with hydrolysis has recently been proposed using a Dictyostelium discoideummyosin construct containing a single tryptophan residue at position 501 in the relay loop, some 3 nm from the active site (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). We demonstrated that tryptophan at this position shows a small quench on nucleotide binding (10–20%) followed by a large enhancement (50–80%) associated with a major conformational change (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar), presumably equivalent to the open-closed transition identified by x-ray crystallography (6Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar). The latter step is freely reversible and slightly favors the open state (low fluorescence M†·ATP) in the presence of ATP under ambient conditions, but coupling to the hydrolysis step pulls the equilibrium over to the more stable M*ADP·Pi state with enhanced fluorescence (5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). Thus, under most conditions, the kinetics of Trp501 enhancement upon interaction with ATP provide a measure of the hydrolysis reaction, in line with earlier schemes (2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar), although relaxation methods reveal these processes to be distinct events (5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, 7Urbanke C. Wray J. Biochem. J. 2001; 358: 165-173Crossref PubMed Scopus (40) Google Scholar). The quench in Trp501 fluorescence that precedes the enhancement is related to the binding process, but details of this step are not easy to characterize because the signal is small and is obscured by the subsequent open-closed transition. In the present study, we have made constructs containing single tryptophan residues near the active site with the aim of probing the initial binding step(s) without any contribution from the conformational changes that are coupled to hydrolysis. The D. discoideum motor domain lacks tryptophan residues near the nucleotide site equivalent to positions Trp113 and Trp131 in vertebrate skeletal myosin. This finding has led to the suggestion that the enhancement seen upon ADP binding to vertebrate skeletal myosin may arise from specific perturbation of the tryptophan residues near the active site (6Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar, 8Kurzawa S. E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar). In skeletal muscle myosin, Trp131 lies in the vicinity of the adenine ring of the bound nucleotide, whereas the Trp113 side chain could be sensitive to nucleotide binding via interaction with the conservative Lys130 residue. We therefore have explored this idea by making the appropriate single tryptophan constructs in the D. discoideum motor domain. We previously showed that substitution of all of the naturally occurring tryptophan residues with phenylalanine in the D. discoideum motor domain had a fairly limited effect on the kinetic pathway (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar), and hence adding back a single tryptophan residue as a site-directed probe at locations throughout the molecule is a potentially viable approach. DISCUSSIONDespite the long history (1Bagshaw C.R. Eccleston J.F. Eckstein F. Goody R.S. Gutfreund H. Trentham D.R. Biochem. J. 1974; 141: 351-364Crossref PubMed Scopus (227) Google Scholar, 2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar, 3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar), the assignment of tryptophan fluorescence changes to specific steps in the vertebrate skeletal muscle myosin and actomyosin ATPase pathways has been controversial and difficult to resolve for a number of reasons. At 20 °C, the rapid isomerization steps are lost within the dead time of the stopped flow apparatus, and only the fluorescence change associated with hydrolysis is clearly resolved. At lower temperatures, additional steps were resolved, which suggested that there are at least two binding isomerizations that are distinct from the hydrolysis step (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar). In the presence of actin, there are further complications arising from the potential of tryptophans in actin to contribute to the signal change, perturbations of myosin tryptophans that are sensitive to actin binding (2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar, 25Yengo C.M. Chrin L. Rovner A.S. Berger C.L. Biochemistry. 1999; 38: 14515-14523Crossref PubMed Scopus (33) Google Scholar), and the technical difficulty of the effect of light scattering changes on the measured fluorescence signal (26Millar N.C. Geeves M.A. Biochem. J. 1988; 249: 735-743Crossref PubMed Scopus (37) Google Scholar). Use of D. discoideum myosin II constructs has the advantage that tryptophan residues can be added or removed by mutagenesis to probe specific regions of the myosin motor domain. Here we focus on the response of tryptophan residues located near the myosin nucleotide binding site. We show that a tryptophan residue at position 129 or 131 responds with similar kinetics (after allowing for species differences) as previously characterized for rabbit skeletal myosin (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar), although the fluorescence change is in the opposite direction. The cause of the fluorescence enhancement on nucleotide binding seen with vertebrate myosins has therefore not been unambiguously identified. There are several candidates for the source of the signal change in vertebrate myosins. First, the two tryptophans in the vicinity of the binding site (Trp113 and Trp131 in rabbit skeletal myosin), which are conserved only among skeletal and cardiac muscle isoforms, are likely to be sensitive to nucleotide binding (6Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar, 8Kurzawa S. E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar), although their local environments may be different from those in D. discoideum myosin to give a net enhancement rather than quench. Park and Burghardt (21Park S. Burghardt T.P. Biochemistry. 2000; 39: 11732-11741Crossref PubMed Scopus (31) Google Scholar) isolated Trp131 emission in skeletal muscle subfragment 1 by means of elimination of its fluorescence contribution using site-specific chemical modification. Their data show that this side chain indeed responds with a ∼20% enhancement upon binding of nucleotide, but they excluded Trp131 as the tryptophan sensitive to ATP hydrolysis on the basis that the signal was similar in different nucleotide states. Based on structural data, Trp113 also cannot be excluded as a potential candidate responsible for nucleotide binding-induced signal changes. The contribution of these residues to the signal change is also indicated by the fact that wild-type D. discoideum myosin II, which lacks Trp131 (and Trp113), does not respond with a significant change in fluorescence on nucleotide binding (8Kurzawa S. E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar, 9Kuhlman P.A. Bagshaw C.R. J. Muscle Res. Cell Motil. 1998; 19: 491-504Crossref PubMed Scopus (36) Google Scholar). The single tryptophan residues of the D. discoideum myosin mutants presented in this study (Trp113, Trp129, and Trp131) were insensitive to the events of the ATPase cycle other than nucleotide binding. However, the combined contributions of Trp113 and Trp131 would lead to a net quench rather than enhancement on nucleotide binding.The conservative tryptophan residue in the relay loop of the motor domain (Trp510 in skeletal, Trp512 in smooth muscle, and Trp501 in D. discoideummyosin), which is sensitive to the open-closed transition, also shows a fluorescence change upon nucleotide binding. Skeletal and smooth muscle myosin relay loop tryptophans show 38 and 30% enhancement upon ADP addition, respectively (21Park S. Burghardt T.P. Biochemistry. 2000; 39: 11732-11741Crossref PubMed Scopus (31) Google Scholar, 22Yengo C.M. Chrin L.R. Rovner A.S. Berger C.L. J. Biol. Chem. 2000; 275: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In the W501+ single tryptophanD. discoideum myosin mutant, a 15–20% quench can be detected on this step (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). It is unlikely that other tryptophans are involved (e.g. the conserved Trp432 in D. discoideum, Trp440 in skeletal myosin) because the W501F and W501Y mutants show almost no fluorescence change upon adding nucleotide (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar,20Batra R. Manstein D.J. Biol. Chem. 1999; 380: 1017-1023Crossref PubMed Scopus (49) Google Scholar). Our data therefore indicate that (i) both the tryptophan(s) near the binding site and the relay loop residue contribute to the fluorescence change upon nucleotide binding prior to the open-closed transition, and (ii) the direction and extent of the fluorescence change of the responsive tryptophans is largely determined by the nonconservative environment surrounding the tryptophan side chain. Thus, we conclude that the global conformational changes undertaken by these myosins are conserved (on the basis of similar rate profiles and functional properties), and even the local movement of particular tryptophan residues could be similar, but the environment experienced by the tryptophan side chains during such movements will vary because of nonconserved residues around them.The acrylamide quenching experiments indicate that a tryptophan residue at position 129 is solvent-exposed, as expected from the crystal structures (17Rayment I. Rypniewski W.R. Schmidt-Bäse K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 50-58Crossref PubMed Scopus (1859) Google Scholar, 18Gulick A.M. Bauer C.B. Thoden J.B. Rayment I. Biochemistry. 1997; 36: 11619-11628Crossref PubMed Scopus (178) Google Scholar). Nevertheless, time-resolved anisotropy measurements indicate that this side chain is not freely mobile, and interconversion between rotamer states occurs on a time scale slower than net rotation of the motor domain, as was also noted for Trp501 (16Málnási-Csizmadia A. Kovács M. Woolley R.J. Botchway S.W. Bagshaw C.R. J. Biol. Chem. 2001; 276: 19483-19490Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Nucleotide binding is accompanied by a protection of Trp129 from collisional quenching by agents in solution, but this residue is now subject to increased static quenching, possibly by direct association with the adenine moiety.Regardless of the specific origin of the fluorescence change, the large quench observed on nucleotide binding to W129+ allows a more precise analysis of the mechanism than has hitherto been possible with wild-type proteins. Furthermore, the signal from Trp129 is not sensitive to the open-closed transition, which removes some of the ambiguity in the analysis. This is particularly important because much of the earlier data exploited analogs such as AMP-PNP and ATPγS to mimic the nonhydrolyzed ATP state, but it is now known that these analogs induce the closed state to a limited extent (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, 7Urbanke C. Wray J. Biochem. J. 2001; 358: 165-173Crossref PubMed Scopus (40) Google Scholar) and hence have a contribution from the signal traditionally assigned to the M*·ADP·Pi state (M**·ADP·Pi in skeletal myosin). ADP has less or no tendency to induce the closed state (5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, 7Urbanke C. Wray J. Biochem. J. 2001; 358: 165-173Crossref PubMed Scopus (40) Google Scholar) and is therefore the simplest nucleotide with which to explore the initial binding steps.In accordance with this concept, the overall profiles of different nucleotides binding to the W129+ construct were similar in character to that observed by Trybus and Taylor (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar) for ADP binding to skeletal myosin at 5 °C (at higher temperatures, the isomerization steps with skeletal myosin are too fast to resolve). The profiles of skeletal andD. discoideum myosins differ in detail in the near loss of the slow phase at high [nucleotide], which indicates that the M†1·L and M†2·L states have nearly the same fluorescence yield in D. discoideum myosin (hence at saturating [nucleotide], step 2b is optically silent). Furthermore, the k obs of the fast phase appeared to extrapolate back to give a large intercept value (250–1000 s−1), whereas it was close to zero in the Trybus and Taylor (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar) data. This aspect is difficult to characterize with accuracy, However, it can be accounted for by an additional weak but competitive nonproductive binding mode of the nucleotide as in ReactionFR2. Here, M#·L might represent a nucleotide bound to the active site in a noncompetent orientation that must dissociate and reassociate (or rearrange while bound) before forming the tightly bound form M†2·L.The fluorescence transients from the double tryptophan construct, W129+/W501+, show a clear distinction between the nucleotide binding steps and the open-closed transition that is coupled to hydrolysis, because the signals have opposite signs (cf. skeletal myosin, where they both give enhancements). Furthermore, the effective hydrolysis rate is slowed to 8 s−1 at 20 °C, which may be useful for developing conformationally resolved single molecule ATPase assays because the transition occurs on the time scale appropriate for video acquisition (27Bagshaw C.R. Conibear P.B. Single Mol. 2000; 1: 269-275Crossref Scopus (4) Google Scholar).Although the aim of this work was to characterize the tryptophan residue(s) in the motor domain that respond to the nucleotide binding steps and to exploit the fluorescence signal to delineate the elementary steps of this process, it is of interest to relate these steps to those that occur in the presence of actin. Previous studies have estimated that the ATP-induced dissociation of acto-D. discoideum motor domain is limited by a first order step of 150–450 s−1 at 20 °C (8Kurzawa S. E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar, 9Kuhlman P.A. Bagshaw C.R. J. Muscle Res. Cell Motil. 1998; 19: 491-504Crossref PubMed Scopus (36) Google Scholar) and is thus comparable with the slower of the two isomerization steps revealed by Trp129 fluorescence. We confirm that the acto-W129+ construct dissociates with a maximum rate constant of 150 s−1 at saturating [ATP]. The conformational changes sensed by the tryptophan residues near the active site (Trp129 and Trp131) are therefore likely to occur before or are coincident with the ATP-induced actomyosin dissociation reaction. This is in agreement with thermodynamic arguments that require coupling with an effectively irreversible step (step 2b with ATP) in the binding mechanism to provide sufficient energy to drive the actomyosin dissociation reaction. We find no evidence of a slower tryptophan fluorescence change sensed by Trp129 after actin dissociation, but previous reports with skeletal myosin where this was observed may reflect a contribution from the open-closed transition (26Millar N.C. Geeves M.A. Biochem. J. 1988; 249: 735-743Crossref PubMed Scopus (37) Google Scholar). Single tryptophan-containing constructs will provide a valuable tool for further delineating communication pathways between the actin site, nucleotide site, and converter region of myosins. Despite the long history (1Bagshaw C.R. Eccleston J.F. Eckstein F. Goody R.S. Gutfreund H. Trentham D.R. Biochem. J. 1974; 141: 351-364Crossref PubMed Scopus (227) Google Scholar, 2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar, 3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar), the assignment of tryptophan fluorescence changes to specific steps in the vertebrate skeletal muscle myosin and actomyosin ATPase pathways has been controversial and difficult to resolve for a number of reasons. At 20 °C, the rapid isomerization steps are lost within the dead time of the stopped flow apparatus, and only the fluorescence change associated with hydrolysis is clearly resolved. At lower temperatures, additional steps were resolved, which suggested that there are at least two binding isomerizations that are distinct from the hydrolysis step (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar). In the presence of actin, there are further complications arising from the potential of tryptophans in actin to contribute to the signal change, perturbations of myosin tryptophans that are sensitive to actin binding (2Johnson K.A. Taylor E.W. Biochemistry. 1978; 17: 3432-3442Crossref PubMed Scopus (167) Google Scholar, 25Yengo C.M. Chrin L. Rovner A.S. Berger C.L. Biochemistry. 1999; 38: 14515-14523Crossref PubMed Scopus (33) Google Scholar), and the technical difficulty of the effect of light scattering changes on the measured fluorescence signal (26Millar N.C. Geeves M.A. Biochem. J. 1988; 249: 735-743Crossref PubMed Scopus (37) Google Scholar). Use of D. discoideum myosin II constructs has the advantage that tryptophan residues can be added or removed by mutagenesis to probe specific regions of the myosin motor domain. Here we focus on the response of tryptophan residues located near the myosin nucleotide binding site. We show that a tryptophan residue at position 129 or 131 responds with similar kinetics (after allowing for species differences) as previously characterized for rabbit skeletal myosin (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar), although the fluorescence change is in the opposite direction. The cause of the fluorescence enhancement on nucleotide binding seen with vertebrate myosins has therefore not been unambiguously identified. There are several candidates for the source of the signal change in vertebrate myosins. First, the two tryptophans in the vicinity of the binding site (Trp113 and Trp131 in rabbit skeletal myosin), which are conserved only among skeletal and cardiac muscle isoforms, are likely to be sensitive to nucleotide binding (6Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar, 8Kurzawa S. E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar), although their local environments may be different from those in D. discoideum myosin to give a net enhancement rather than quench. Park and Burghardt (21Park S. Burghardt T.P. Biochemistry. 2000; 39: 11732-11741Crossref PubMed Scopus (31) Google Scholar) isolated Trp131 emission in skeletal muscle subfragment 1 by means of elimination of its fluorescence contribution using site-specific chemical modification. Their data show that this side chain indeed responds with a ∼20% enhancement upon binding of nucleotide, but they excluded Trp131 as the tryptophan sensitive to ATP hydrolysis on the basis that the signal was similar in different nucleotide states. Based on structural data, Trp113 also cannot be excluded as a potential candidate responsible for nucleotide binding-induced signal changes. The contribution of these residues to the signal change is also indicated by the fact that wild-type D. discoideum myosin II, which lacks Trp131 (and Trp113), does not respond with a significant change in fluorescence on nucleotide binding (8Kurzawa S. E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar, 9Kuhlman P.A. Bagshaw C.R. J. Muscle Res. Cell Motil. 1998; 19: 491-504Crossref PubMed Scopus (36) Google Scholar). The single tryptophan residues of the D. discoideum myosin mutants presented in this study (Trp113, Trp129, and Trp131) were insensitive to the events of the ATPase cycle other than nucleotide binding. However, the combined contributions of Trp113 and Trp131 would lead to a net quench rather than enhancement on nucleotide binding. The conservative tryptophan residue in the relay loop of the motor domain (Trp510 in skeletal, Trp512 in smooth muscle, and Trp501 in D. discoideummyosin), which is sensitive to the open-closed transition, also shows a fluorescence change upon nucleotide binding. Skeletal and smooth muscle myosin relay loop tryptophans show 38 and 30% enhancement upon ADP addition, respectively (21Park S. Burghardt T.P. Biochemistry. 2000; 39: 11732-11741Crossref PubMed Scopus (31) Google Scholar, 22Yengo C.M. Chrin L.R. Rovner A.S. Berger C.L. J. Biol. Chem. 2000; 275: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In the W501+ single tryptophanD. discoideum myosin mutant, a 15–20% quench can be detected on this step (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). It is unlikely that other tryptophans are involved (e.g. the conserved Trp432 in D. discoideum, Trp440 in skeletal myosin) because the W501F and W501Y mutants show almost no fluorescence change upon adding nucleotide (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar,20Batra R. Manstein D.J. Biol. Chem. 1999; 380: 1017-1023Crossref PubMed Scopus (49) Google Scholar). Our data therefore indicate that (i) both the tryptophan(s) near the binding site and the relay loop residue contribute to the fluorescence change upon nucleotide binding prior to the open-closed transition, and (ii) the direction and extent of the fluorescence change of the responsive tryptophans is largely determined by the nonconservative environment surrounding the tryptophan side chain. Thus, we conclude that the global conformational changes undertaken by these myosins are conserved (on the basis of similar rate profiles and functional properties), and even the local movement of particular tryptophan residues could be similar, but the environment experienced by the tryptophan side chains during such movements will vary because of nonconserved residues around them. The acrylamide quenching experiments indicate that a tryptophan residue at position 129 is solvent-exposed, as expected from the crystal structures (17Rayment I. Rypniewski W.R. Schmidt-Bäse K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 50-58Crossref PubMed Scopus (1859) Google Scholar, 18Gulick A.M. Bauer C.B. Thoden J.B. Rayment I. Biochemistry. 1997; 36: 11619-11628Crossref PubMed Scopus (178) Google Scholar). Nevertheless, time-resolved anisotropy measurements indicate that this side chain is not freely mobile, and interconversion between rotamer states occurs on a time scale slower than net rotation of the motor domain, as was also noted for Trp501 (16Málnási-Csizmadia A. Kovács M. Woolley R.J. Botchway S.W. Bagshaw C.R. J. Biol. Chem. 2001; 276: 19483-19490Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Nucleotide binding is accompanied by a protection of Trp129 from collisional quenching by agents in solution, but this residue is now subject to increased static quenching, possibly by direct association with the adenine moiety. Regardless of the specific origin of the fluorescence change, the large quench observed on nucleotide binding to W129+ allows a more precise analysis of the mechanism than has hitherto been possible with wild-type proteins. Furthermore, the signal from Trp129 is not sensitive to the open-closed transition, which removes some of the ambiguity in the analysis. This is particularly important because much of the earlier data exploited analogs such as AMP-PNP and ATPγS to mimic the nonhydrolyzed ATP state, but it is now known that these analogs induce the closed state to a limited extent (4Málnási-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, 7Urbanke C. Wray J. Biochem. J. 2001; 358: 165-173Crossref PubMed Scopus (40) Google Scholar) and hence have a contribution from the signal traditionally assigned to the M*·ADP·Pi state (M**·ADP·Pi in skeletal myosin). ADP has less or no tendency to induce the closed state (5Málnási-Csizmadia A. Pearson D.S. Kovács M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, 7Urbanke C. Wray J. Biochem. J. 2001; 358: 165-173Crossref PubMed Scopus (40) Google Scholar) and is therefore the simplest nucleotide with which to explore the initial binding steps. In accordance with this concept, the overall profiles of different nucleotides binding to the W129+ construct were similar in character to that observed by Trybus and Taylor (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar) for ADP binding to skeletal myosin at 5 °C (at higher temperatures, the isomerization steps with skeletal myosin are too fast to resolve). The profiles of skeletal andD. discoideum myosins differ in detail in the near loss of the slow phase at high [nucleotide], which indicates that the M†1·L and M†2·L states have nearly the same fluorescence yield in D. discoideum myosin (hence at saturating [nucleotide], step 2b is optically silent). Furthermore, the k obs of the fast phase appeared to extrapolate back to give a large intercept value (250–1000 s−1), whereas it was close to zero in the Trybus and Taylor (3Trybus K.M. Taylor E.W. Biochemistry. 1982; 21: 1284-1294Crossref PubMed Scopus (72) Google Scholar) data. This aspect is difficult to characterize with accuracy, However, it can be accounted for by an additional weak but competitive nonproductive binding mode of the nucleotide as in ReactionFR2. Here, M#·L might represent a nucleotide bound to the active site in a noncompetent orientation that must dissociate and reassociate (or rearrange while bound) before forming the tightly bound form M†2·L. The fluorescence transients from the double tryptophan construct, W129+/W501+, show a clear distinction between the nucleotide binding steps and the open-closed transition that is coupled to hydrolysis, because the signals have opposite signs (cf. skeletal myosin, where they both give enhancements). Furthermore, the effective hydrolysis rate is slowed to 8 s−1 at 20 °C, which may be useful for developing conformationally resolved single molecule ATPase assays because the transition occurs on the time scale appropriate for video acquisition (27Bagshaw C.R. Conibear P.B. Single Mol. 2000; 1: 269-275Crossref Scopus (4) Google Scholar). Although the aim of this work was to characterize the tryptophan residue(s) in the motor domain that respond to the nucleotide binding steps and to exploit the fluorescence signal to delineate the elementary steps of this process, it is of interest to relate these steps to those that occur in the presence of actin. Previous studies have estimated that the ATP-induced dissociation of acto-D. discoideum motor domain is limited by a first order step of 150–450 s−1 at 20 °C (8Kurzawa S. E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar, 9Kuhlman P.A. Bagshaw C.R. J. Muscle Res. Cell Motil. 1998; 19: 491-504Crossref PubMed Scopus (36) Google Scholar) and is thus comparable with the slower of the two isomerization steps revealed by Trp129 fluorescence. We confirm that the acto-W129+ construct dissociates with a maximum rate constant of 150 s−1 at saturating [ATP]. The conformational changes sensed by the tryptophan residues near the active site (Trp129 and Trp131) are therefore likely to occur before or are coincident with the ATP-induced actomyosin dissociation reaction. This is in agreement with thermodynamic arguments that require coupling with an effectively irreversible step (step 2b with ATP) in the binding mechanism to provide sufficient energy to drive the actomyosin dissociation reaction. We find no evidence of a slower tryptophan fluorescence change sensed by Trp129 after actin dissociation, but previous reports with skeletal myosin where this was observed may reflect a contribution from the open-closed transition (26Millar N.C. Geeves M.A. Biochem. J. 1988; 249: 735-743Crossref PubMed Scopus (37) Google Scholar). Single tryptophan-containing constructs will provide a valuable tool for further delineating communication pathways between the actin site, nucleotide site, and converter region of myosins. We thank Dr. Stanley R. Botchway (Rutherford Appleton Laboratory) for assistance in conducting the time-correlated single photon counting measurements." @default.
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W1990804119 title "Analysis of Nucleotide Binding to DictyosteliumMyosin II Motor Domains Containing a Single Tryptophan Near the Active Site" @default.
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