Magnesium, ADP, and actin binding linkage of myosin V: evidence for multiple myosin V-ADP and actomyosin V-ADP states

The [Mg(2+)] dependence of ADP binding to myosin V and actomyosin V was measured from the fluorescence of mantADP. Time courses of MgmantADP dissociation from myosin V and actomyosin V are biphasic with fast observed rate constants that depend on the [Mg(2+)] and slow observed rate constants that are [Mg(2+)]-independent. Two myosin V-MgADP states that are in reversible equilibrium, one that exchanges nucleotide and cation slowly (strong binding) and one that exchanges nucleotide and cation rapidly (weak binding), account for the data. The two myosin V-MgADP states are of comparable energies, as indicated by the relatively equimolar partitioning at saturating magnesium. Actin binding lowers the affinity for bound Mg(2+) 2-fold but shifts the isomerization equilibrium approximately 6-fold to the weak ADP binding state, lowering the affinity and accelerating the overall rate of MgADP release. Actin does not weaken the affinity or accelerate the release of cation-free ADP, indicating that actin and ADP binding linkage is magnesium-dependent. Myosin V and myosin V-ADP binding to actin was assayed from the quenching of pyrene actin fluorescence. Time courses of myosin V-ADP binding and release are biphasic, consistent with the existence of two (weak and strong) quenched pyrene actomyosin V-ADP conformations. We favor a sequential mechanism for actomyosin V dissociation with a transition from strong to weak actin-binding conformations preceding dissociation. The data provide evidence for multiple myosin-ADP and actomyosin-ADP states and establish a kinetic and thermodynamic framework for defining the magnesium-dependent coupling between the actin and nucleotide binding sites of myosin.     

The calcium binding properties of phosphorylated and unphosphorylated cardiac and skeletal myosins.

Porcine left ventricular cardiac myosin and rabbit white skeletal myosin were phosphorylated by rabbit skeletal myosin light chain kinase and their Ca2+ binding properties were examined by equilibrium dialysis techniques. No significant effect of phosphorylation on the Ca2+ binding properties of these myosins was observed. Both types of striated muscle myosins bound approximately 2 mol of Ca2+/mol of myosin with similar affinities of 3 x 10(7) M-1. In the presence of 3 x 10(-4) M Mg2+ the myosins bound Ca2+ with a reduced affinity of 3 to 4 x 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the binding sites on myosin, the changes in Ca2+ binding can be accounted for by a Mg2+ affinity of 2.5 to 3.0 x 10(5) M-1.     

Strong binding of myosin heads stretches and twists the actin helix

Calculation of the size of the power stroke of the myosin motor in contracting muscle requires knowledge of the compliance of the myofilaments. Current estimates of actin compliance vary significantly introducing uncertainty in the mechanical parameters of the motor. Using x-ray diffraction on small bundles of permeabilized fibers from rabbit muscle we show that strong binding of myosin heads changes directly the actin helix. The spacing of the 2.73-nm meridional x-ray reflection increased by 0.22% when relaxed fibers were put into low-tension rigor (<10 kN/m(2)) demonstrating that strongly bound myosin heads elongate the actin filaments even in the absence of external tension. The pitch of the 5.9-nm actin layer line increased by approximately 0.62% and that of the 5.1-nm layer line decreased by approximately 0.26%, suggesting that the elongation is accompanied by a decrease in its helical angle (approximately 166 degrees) by approximately 0.8 degrees. This effect explains the difference between actin compliance revealed from mechanical experiments with single fibers and from x-ray diffraction on whole muscles. Our measurement of actin compliance obtained by applying tension to fibers in rigor is consistent with the results of mechanical measurements.     

The myosin relay helix to converter interface remains intact throughout the actomyosin ATPase cycle

Crystal structures of the myosin motor domain in the presence of different nucleotides show the lever arm domain in two basic angular states, postulated to represent prestroke and poststroke states, respectively (Rayment, I. (1996) J. Biol. Chem. 271, 15850-15853; Dominguez, R., Freyzon, Y., Trybus, K. M., and Cohen, C. (1998) Cell 94, 559-571). Contact is maintained between two domains, the relay and the converter, in both of these angular states. Therefore it has been proposed by Dominguez et al. (cited above) that this contact is critical for mechanically driving the angular change of the lever arm domain. However, structural information is lacking on whether this contact is maintained throughout the actin-activated myosin ATPase cycle. To test the functional importance of this interdomain contact, we introduced cysteines into the sequence of a "cysteine-light" myosin motor at position 499 on the lower cleft and position 738 on the converter domain (Shih, W. M., Gryczynski, Z., Lakowicz, J. L., and Spudich, J. A. (2000) Cell 102, 683-694). Disulfide cross-linking could be induced. The cross-link had minimal effects on actin binding, ATP-induced actin release, and actin-activated ATPase. These results demonstrate that the relay/converter interface remains intact in the actin strongly bound state of myosin and throughout the entire actin-activated myosin ATPase cycle.     

A hydrophobic region on myosin light chains modulated by divalent cations

A hydrophobic region was detected on several types of myosin light chain by enhancement of the quantum yield of 1-anilino-8-naphthalenesulfonate (ANS) fluorescence. The character of this non-polar region was altered by the binding of Ca2+ or Mg2+ to the light chain, the quantum yield of the ANS being increased, and its emission maximum undergoing a blue-shift. These changes enabled the binding of divalent cations to the myosin light chains to be monitored. When Ca2+ was bound to gizzard regulatory light chain, a biphasic enhancement of light-chain-bound ANS fluorescence occurred, the first phase taking place in the micromolar range and the second in the millimolar range of free Ca2+ concentration. Enhancement of protein-bound ANS fluorescence as divalent cations were bound was also observed with other types of myosin light chain.     

Linear dichroism of acrylodan-labeled tropomyosin and myosin subfragment 1 bound to actin in myofibrils.

Muscle contraction can be activated by the binding of myosin heads to the thin filament, which appears to result in thin filament structural changes. In vitro studies of reconstituted muscle thin filaments have shown changes in tropomyosin-actin geometry associated with the binding of myosin subfragment 1 to actin. Further information about these structural changes was obtained with fluorescence-detected linear dichroism of tropomyosin, which was labeled at Cys 190 with acrylodan and incorporated into oriented ghost myofibrils. The fluorescence from three sarcomeres of the fibril was collected with the high numerical aperture objective of a microscope and the dichroic ratio, R (0/90 degrees), for excitation parallel/perpendicular to the fibril, was obtained, which gave the average probe dipole polar angle, Theta. For both acrylodan-labeled tropomyosin bound to actin in fibrils and in Mg2+ paracrystals, Theta congruent to 52 degrees +/- 1.0 degrees, allowing for a small degree of orientational disorder. Binding of myosin subfragment 1 to actin in fibrils did not change Theta; i.e., the orientation of the rigidly bound probe on tropomyosin did not change relative to the actin axis. These data indicate that myosin subfragment 1 binding to actin does not appreciably perturb the structure of tropomyosin near the probe and suggest that the geometry changes are such as to maintain the parallel orientation of the tropomyosin and actin axes, a finding consistent with models of muscle regulation. Data are also presented for effects of MgADP on the orientation of labeled myosin subfragment 1 bound to actin in myofibrils.     

Actin-induced closure of the actin-binding cleft of smooth muscle myosin

The putative actin-binding interface of myosin is separated by a large cleft that extends into the base of the nucleotide binding pocket, suggesting that it may be important for mediating the nucleotide-dependent changes in the affinity for myosin on actin. We have genetically engineered a truncated version of smooth muscle myosin containing the motor domain and the essential light chain-binding region (MDE), with a single tryptophan residue at position 425 (F425W-MDE) in the actin-binding cleft. Steady-state fluorescence of F425W-MDE demonstrates that Trp-425 is in a more solvent-exposed conformation in the presence of MgATP than in the presence of MgADP or absence of nucleotide, consistent with closure of the actin-binding cleft in the strongly bound states of MgATPase cycle for myosin. Transient kinetic experiments demonstrate a direct correlation between the rates of strong actin binding and the conformation of Trp-425 in the actin-binding cleft, and suggest the existence of a novel conformation of myosin not previously seen in solution or by x-ray crystallography. Thus, these results directly demonstrate that: 1) the conformation of the actin-binding cleft mediates the affinity of myosin for actin in a nucleotide-dependent manner, and 2) actin induces conformational changes in myosin required to generate force and motion during muscle contraction.     

Characterization of an actin flow artifact that occurs during the presteady state dissociation of actosubfragment-1

Models for the activation of the myosin subfragment-1 (S-1) ATPase activity by actin describe transitions that occur between kinetic intermediate states during steady state hydrolysis of ATP. These states consist of myosin-nucleotide complexes in rapid equilibrium binding with actin, but steady state measurements of actin binding during hydrolysis lead only to a weighted average of the individual binding constants involved. In the current work, in order to determine the individual binding constants involved in the activation process, we have investigated the presteady state kinetics of the dissociation of actomyosin by ATP. We find that an actin flow artifact appears to dominate the time course of dissociation, and characterization of this artifact reveals that its magnitude rises linearly (approximately) with the concentration of bound S-1. Attempts to subtract the actin flow artifact from the actoS-1 dissociation signal were not entirely successful due at least partially to the transient nature of the bound S-1 concentration in the first few milliseconds. However, further studies reveal that if the order of addition of actin, ATP, and S-1 are varied, the observed light scattering transients are essentially superimposable. One possible explanation of these data is that the binding constants for myosin-ATP and myosin-ADP-Pi to actin are equal. However, it is also possible that the flow artifact is so large that further analysis is precluded. In addition, we show that the actin flow artifact has little effect on the fluorescence measurements of the phosphate burst reported previously. Therefore, the prior interpretation of the fluorescence data remains unchanged.     

The effects of calcium and magnesium ions on the adenosine triphosphatase and inosine triphosphatase activities of myosin A

1. The effects of Ca(2+) and Mg(2+) on the enzymic activity of myosin were studied with myosin preparations treated by the ion-exchange resin Chelex-100. A reaction mixture containing 0.05m-potassium chloride was chosen in which the effects of univalent ions such as K(+), Na(+) and Cl(-) do not change significantly with small variations in their concentrations. 2. The relationship between the rate of hydrolysis of ATP or ITP and the concentration of Ca(2+) suggests that a relatively weak binding of Ca(2+) either to myosin or to the substrate nucleotide is responsible for the activation of the enzymic activity. According to the experiments with an ultrafiltration technique, the binding of Ca(2+) to myosin proceeds in at least two steps, the first occurring at one site on every 500000 atomic mass units of myosin with an apparent association constant, K(app.), 1.3x10(6)m(-1), and the second seeming to be so weak that its binding parameters cannot be determined by the method used. The first type of Ca(2+) binding is not observable with N-ethylmaleimide-modified myosin, yet this modified myosin shows activation by Ca(2+) of its adenosine triphosphatase and inosine triphosphatase. 3. The inhibition by Mg(2+) can be related to a binding reaction of Mg(2+) with myosin having K(app.) approximately 10(6)m(-1). Mg(2+) replaces the Ca(2+) bound tightly to myosin. The K(app.) for Mg(2+)-myosin binding calculated by assuming a competition between Ca(2+) and Mg(2+) for the same site is 2.1x10(5)-3.0x10(5)m(-1). When myosin is modified with a thiol reagent (p-mercuribenzoate) at a certain ratio to myosin, the inhibition by Mg(2+) becomes unobservable. 4. The behaviour of the hydrolytic activity of myosin on ATP or ITP in the presence of both Ca(2+) and Mg(2+) is consistent with the explanation that the inhibition by Mg(2+) is due to the tight binding of Mg(2+) to myosin, whereas the activation by Ca(2+) is caused either by a weak binding of Ca(2+) to myosin or by CaATP(2-) or by both.     

The structural basis of myosin V processive movement as revealed by electron cryomicroscopy

The processive motor myosin V has a relatively high affinity for actin in the presence of ATP and, thus, offers the unique opportunity to visualize some of the weaker, hitherto inaccessible, actin bound states of the ATPase cycle. Here, electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional (3D) reconstructions provide the atomic models of myosin V in both weak and strong actin bound states. One structure shows that ATP binding opens the long cleft dividing the actin binding region of the motor domain, thus destroying the strong binding actomyosin interface while rearranging loop 2 as a tether. Nucleotide analogs showed a second new state in which the lever arm points upward, in a prepower-stroke configuration (lever arm up) bound to actin before phosphate release. Our findings reveal how the structural elements of myosin V work together to allow myosin V to step along actin for multiple ATPase cycles without dissociating.     

Crystal structure of the motor domain of a class-I myosin

The crystal structure of the motor domain of Dictyostelium discoideum myosin-IE, a monomeric unconventional myosin, was determined. The crystallographic asymmetric unit contains four independently resolved molecules, highlighting regions that undergo large conformational changes. Differences are particularly pronounced in the actin binding region and the converter domain. The changes in position of the converter domain reflect movements both parallel to and perpendicular to the actin axis. The orientation of the converter domain is approximately 30 degrees further up than in other myosin structures, indicating that MyoE can produce a larger power stroke by rotating its lever arm through a larger angle. The role of extended loops near the actin-binding site is discussed in the context of cellular localization. The core regions of the motor domain are similar, and the structure reveals how that core is stabilized in the absence of an N-terminal SH3-like domain.     

Direct visualization by electron microscopy of the weakly bound intermediates in the actomyosin adenosine triphosphatase cycle.

We used a novel stopped-flow/rapid-freezing machine to prepare the transient intermediates in the actin-myosin adenosine triphosphatase (ATPase) cycle for direct observation by electron microscopy. We focused on the low affinity complexes of myosin-adenosine triphosphate (ATP) and myosin-adenosine diphosphate (ADP)-Pi with actin filaments since the transition from these states to the high affinity actin-myosin-ADP and actin-myosin states is postulated to generate the molecular motion that drives muscle contraction and other types of cellular movements. After rapid freezing and metal replication of mixtures of myosin subfragment-1, actin filaments, and ATP, the structure of the weakly bound intermediates is indistinguishable from nucleotide-free rigor complexes. In particular, the average angle of attachment of the myosin head to the actin filament is approximately 40 degrees in both cases. At all stages in the ATPase cycle, the configuration of most of the myosin heads bound to actin filaments is similar, and the part of the myosin head preserved in freeze-fracture replicas does not tilt by more than a few degrees during the transition from the low affinity to high affinity states. In contrast, myosin heads chemically cross-linked to actin filaments differ in their attachment angles from ordered at 40 degrees without ATP to nearly random in the presence of ATP when viewed by negative staining (Craig, R., L.E. Greene, and E. Eisenberg. 1985. Proc. Natl. Acad. Sci. USA. 82:3247-3251, and confirmed here), freezing in vitreous ice (Applegate, D., and P. Flicker. 1987. J. Biol. Chem. 262:6856-6863), and in replicas of rapidly frozen samples. This suggests that many of the cross-linked heads in these preparations are dissociated from but tethered to the actin filaments in the presence of ATP. These observations suggest that the molecular motion produced by myosin and actin takes place with the myosin head at a point some distance from the actin binding site or does not involve a large change in the shape of the myosin head.     

Influence of trace amount of calponin on smooth muscle myosin in different states

The smooth muscle contraction and relaxation areprimarily regulated by the reversible Ca2 -calmodulin(CaM) dependent phosphorylation of myosin light chaincatalyzed by myosin light chain kinase (MLCK) [1–5].However, the detailed aspects of the regulation …     

Binding of caldesmon to smooth muscle myosin

Caldesmon, a major calmodulin binding protein, was found to bind smooth muscle myosin. Addition of caldesmon to smooth muscle myosin induced the formation of small aggregates of myosin in the absence of Ca2+-calmodulin, but not in the presence of Ca2+-calmodulin. The binding site of myosin was studied by using caldesmon-Sepharose 4B affinity chromatography. Subfragment 1 was not retained by the column, while heavy meromyosin and subfragment 2 were bound to the caldesmon affinity column in the absence of Ca2+-calmodulin but not in its presence. It was therefore concluded that the binding site of caldesmon on myosin molecule was the subfragment 2 region and that binding of caldesmon to myosin was abolished in the presence of Ca2+ and calmodulin. Cross-linking of actin and myosin mediated by caldesmon was studied. While actomyosin was completely dissociated in the presence of Mg2+-ATP, the addition of caldesmon caused aggregation of the actomyosin. By low speed centrifugation at which actomyosin alone was not precipitated in the presence of Mg2+-ATP, the aggregate induced by caldesmon was precipitated and the composition of the precipitate was found to be actin, caldesmon, and myosin. In the presence of Mg2+-ATP, pure actin did not bind to a myosin-Sepharose 4B affinity column, while all of the actin was retained when the actin/caldesmon mixture was applied to the column. These results indicate that caldesmon can cross-link actin and myosin.     

An immunological approach to myosin light-chain function in thick filament linked regulation. 2. Effects of anti-scallop myosin light-chain antibodies. Possible regulatory role for the essential light chain

Specific antibodies directed against the regulatory light chains (R-LC) or essential light chains (SH-LC) of scallop myosin abolished calcium regulation in myofibrils, myosin, and heavy meromyosin by elevating the actin-activated Mg2+-ATPase activity in the absence of calcium. Calcium dependence was completely eliminated at molar ratios of 2.5-3 antibodies bound per myosin. Monovalent anti-R-LC Fab and anti-SH-LC Fab fragments also desensitized myofibrils fully. High Ca2+-ATPase activity remained unaffected by the antibodies. Anti-SH-LC IgG reduced to about one-half the actin-activated Mg2+-ATPase in the presence of calcium and the potassium-activated ethylenediaminetetraacetic acid (EDTA)-ATPase activities. Anti-SH-LC Fab, however, desensitized without inhibiting the actin-activated Mg2+-ATPase. The desensitizing effect of both antibodies was abolished by prior absorption with the homologous myosin light chain. Calcium binding and R-LC and anti-SH-LC IgG's and by anti-SH-LC Fab. The anti-R-LC Fab fragment induced a significant (70%) dissociation of R-LC from myofibrils and myosins with concomitant losses in calcium binding. In contrast, anti-R-LC IgG prevented the dissociation of R-LC from myosin by EDTA. Binding of anti-R-LC IgG to myofibrils was proportional to thier R-LC content. Increased amounts of anti-SH-LC IgG were bound by myofibrils devoid of R-LC. Bound anti-SH-LC antibody significantly inhibited the reuptake of R-LC by EDTA-treated myofibrils as well as the full binding of anti-R-LC antibody. Certain rabbits produced a population of anti-SH-LC antibodies which were specific for this light chain and bound extensively to myosin but failed to desensitize it (nondesensitizing anti-SH-LC antibody). The desensitizing and nondesensitizing anti-SH-LC populations bound to different regions of the SH-LC on the myosin, and the binding of the two types of antibody to the SH-LC was nearly additive. The nondesensitizing SH-antibody inhibited the reuptake of R-LC less, and its binding to myofibrils was not influenced by the absence of R-LC. These studies indicate a direct or indirect involvement of the SH-LC's in myosin-linked regulation, raise the possibility of an interaction between the R-LC and SH-LC, and confirm the regulatory function of the scallop R-LC. A model for a relative location of the two types of light chains and the involvement of the subfragment-2 region of myosin linked regulation is discussed.     

Kinetic characterization of the weak binding states of myosin V

Myosin V is a molecular motor shown to move processively along actin filaments. We investigated the properties of the weak binding states of monomeric myosin V containing a single IQ domain (MV 1IQ) to determine if the affinities of these states are increased as compared to conventional myosin. Further, using a combination of non-hydrolyzable nucleotide analogues and mutations that block ATP hydrolysis, we sought to probe the states that are populated during ATP-induced dissociation of actomyosin. MV 1IQ binds actin with a K(d) = 4 microM in the presence of ATP gamma S at 50 mM KCl, which is 10-20-fold tighter than that of nonprocessive class II myosins. Mutations within the switch II region trapped MV 1IQ in two distinct M.ATP states with very different actin binding affinities (K(d) = 0.2 and 2 microM). Actin binding may change the conformation of the switch II region, suggesting that elements of the nucleotide binding pocket will be in a different conformation when bound to actin than is seen in any of the myosin crystal structures to date.     

Backward movements of cross-bridges by application of stretch and by binding of MgADP to skeletal muscle fibers in the rigor state as studied by x-ray diffraction

The effects of the applied stretch and MgADP binding on the structure of the actomyosin cross-bridges in rabbit and/or frog skeletal muscle fibers in the rigor state have been investigated with improved resolution by x-ray diffraction using synchrotron radiation. The results showed a remarkable structural similarity between cross-bridge states induced by stretch and MgADP binding. The intensities of the 14.4- and 7.2-nm meridional reflections increased by approximately 23 and 47%, respectively, when 1 mM MgADP was added to the rigor rabbit muscle fibers in the presence of ATP-depletion backup system and an inhibitor for muscle adenylate kinase or by approximately 33 and 17%, respectively, when rigor frog muscle was stretched by approximately 4.5% of the initial muscle length. In addition, both MgADP binding and stretch induced a small but genuine intensity decrease in the region close to the meridian of the 5.9-nm layer line while retaining the intensity profile of its outer portion. No appreciable influence was observed in the intensities of the higher order meridional reflections of the 14.4-nm repeat and the other actin-based reflections as well as the equatorial reflections, indicating a lack of detachment of cross-bridges in both cases. The changes in the axial spacings of the actin-based and the 14.4-nm-based reflections were observed and associated with the tension change. These results indicate that stretch and ADP binding mediate similar structural changes, being in the correct direction to those expected for that the conformational changes are induced in the outer portion distant from the catalytic domain of attached cross-bridges. Modeling of conformational changes of the attached myosin head suggested a small but significant movement (about 10-20 degrees) in the light chain-binding domain of the head toward the M-line of the sarcomere. Both chemical (ADP binding) and mechanical (stretch) intervensions can reverse the contractile cycle by causing a backward movement of this domain of attached myosin heads in the rigor state.     

Argininamide binding arrests global motions in HIV-1 TAR RNA: comparison with Mg2+-induced conformational stabilization

The structure and dynamics of the stem-loop transactivation response element (TAR) RNA from the human immunodeficiency virus type-1 (HIV-1) bound to the ligand argininamide (ARG) has been characterized using a combination of a large number of residual dipolar couplings (RDCs) and trans-hydrogen bond NMR methodology. Binding of ARG to TAR changes the average inter-helical angle between the two stems from approximately 47 degrees in the free state to approximately 11 degrees in the bound state, and leads to the arrest of large amplitude (+/-46 degrees ) inter-helical motions observed previously in the free state. While the global structural dynamics of TAR-ARG is similar to that previously reported for TAR bound to Mg2+, there are substantial differences in the hydrogen bond alignment of bulge and neighboring residues. Based on a novel H5(C5)NN experiment for probing hydrogen-mediated 2hJ(N,N) scalar couplings as well as measured RDCs, the TAR-ARG complex is stabilized by a U38-A27.U23 base-triple involving an A27.U23 reverse Hoogsteen hydrogen bond alignment as well as by a A22-U40 Watson-Crick base-pair at the junction of stem I. These hydrogen bond alignments are not observed in either the free or Mg2+ bound forms of TAR. The combined conformational analysis of TAR under three states reveals that ligands and divalent ions can stabilize similar RNA global conformations through distinct interactions involving different hydrogen bond alignments in the RNA.     

Studies of the interaction between titin and myosin

The interaction of titin with myosin has been studied by binding assays and electron microscopy. Electron micrographs of the titin-myosin complex suggest a binding site near the tip of the tail of the myosin molecule. The distance from the myosin head-tail junction to titin indicates binding 20-30 nm from the myosin COOH terminus. Consistent with this, micrographs of titin-light meromyosin (LMM) show binding near the end of the LMM molecule. Plots of myosin- and LMM-attachment positions along the titin molecule show binding predominantly in the region located in the A band in situ, which is consistent with the proposal that titin regulates thick filament assembly. Estimates of the apparent dissociation constant of the titin-LMM complex were approximately 20 nM. Assays of LMM cyanogen bromide fragments also suggested a strong binding site near the COOH terminus. Proteolysis of a COOH-terminal 17.6-kD CNBr fragment isolated from whole myosin resulted in eight peptides of which only one, comprising 17 residues, bound strongly to titin. Two isoforms of this peptide were detected by protein sequencing. Similar binding data were obtained using synthetic versions of both isoforms. The peptide is located immediately COOH- terminal of the fourth "skip" residue in the myosin tail, which is consistent with the electron microscopy. Skip-4 may have a role in determining thick filament structure, by allowing abrupt bending of the myosin tail close to the titin-binding site.     

Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding.

We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment.