Sengen Xu

The M·ADP·Pi State Is Required for Helical Order in the Thick Filaments of Skeletal Muscle

The thick filaments of mammalian and avian skeletal muscle fibers are disordered at low temperature, but become increasingly ordered into an helical structure as the temperature is raised. Wray and colleagues (Schlichting, I., and J. Wray. 1986. J. Muscle Res. Cell Motil. 7:79; Wray, J., R. S. Goody, and K. Holmes. 1986. Adv. Exp. Med. Biol. 226:49-59) interpreted the transition as reflecting a coupling between nucleotide state and global conformation with M.ATP (disordered) being favored at 0 degrees C and M.ADP.P(i) (ordered) at 20 degrees C. However, hitherto this has been limited to a qualitative correlation and the biochemical state of the myosin heads required to obtain the helical array has not been unequivocally identified. In the present study we have critically tested whether the helical arrangement of the myosin heads requires the M.ADP.P(i) state. X-ray diffraction patterns were recorded from skinned rabbit psoas muscle fiber bundles stretched to non-overlap to avoid complications due to interaction with actin. The effect of temperature on the intensities of the myosin-based layer lines and on the phosphate burst of myosin hydrolyzing ATP in solution were examined under closely matched conditions. The results showed that the fraction of myosin mass in the helix closely followed that of the fraction of myosin in the M.ADP.P(i) state. Similar results were found by using a series of nucleoside triphosphates, including CTP and GTP. In addition, fibers treated by N-phenylmaleimide (Barnett, V. A., A. Ehrlich, and M. Schoenberg. 1992. Biophys. J. 61:358-367) so that the myosin was exclusively in the M.ATP state revealed no helical order. Diffraction patterns from muscle fibers in nucleotide-free and in ADP-containing solutions did not show helical structure. All these confirmed that in the presence of nucleotides, the M.NDP.P(i) state is required for helical order. We also found that the spacing of the third meridional reflection of the thick filament is linked to the helical order. The spacing in the ordered M.NDP.P(i) state is 143.4 A, but in the disordered state, it is 144. 2 A. This may be explained by the different interference functions for the myosin heads and the thick filament backbone.

Temperature-induced structural changes in the myosin thick filament of skinned rabbit psoas muscle.

By using synchrotron radiation and an imaging plate for recording diffraction patterns, we have obtained high-resolution x-ray patterns from relaxed rabbit psoas muscle at temperatures ranging from 1 degree C to 30 degrees C. This allowed us to obtain intensity profiles of the first six myosin layer lines and apply a model-building approach for structural analysis. At temperatures 20 degrees C and higher, the layer lines are sharp with clearly defined maxima. Modeling based on the data obtained at 20 degrees C reveals that the average center of the cross-bridges is at 135 A from the center of the thick filament and both of the myosin heads appear to wrap around the backbone. At 10 degrees C and lower, the layer lines become very weak and diffuse scattering increases considerably. At 4 degrees C, the peak of the first layer line shifts toward the meridian from 0.0047 to 0.0038 A(-1) and decreases in intensity approximately by a factor of four compared to that at 20 degrees C, although the intensities of higher-order layer lines remain approximately 10-15% of the first layer line. Our modeling suggests that as the temperature is lowered from 20 degrees C to 4 degrees C the center of cross-bridges extends radially away from the center of the filament (135 A to 175 A). Furthermore, the fraction of helically ordered cross-bridges decreases at least by a factor of two, while the isotropic disorder (the temperature factor) remains approximately unchanged. Our results on the order/disordering effects of temperature are in general agreement with earlier results of Wray [Wray, J. 1987. Structure of relaxed myosin filaments in relation to nucleotide state in vertebrate skeletal muscle. J. Muscle Res. Cell Motil. 8:62a (Abstr.)] and Lowy et al. (Lowy, J., D. Popp, and A. A. Stewart. 1991. X-ray studies of order-disorder transitions in the myosin heads of skinned rabbit psoas muscles. Biophys. J. 60:812-824). and support Poulsen and Lowys hypothesis of coexistence of ordered and disordered cross-bridge populations in muscle (Poulsen, F. R., and J. Lowy. 1983. Small angle scattering from myosin heads in relaxed and rigor frog skeletal muscle. Nature (Lond.). 303:146-152.). However, our results added new insights into the disordered population. Present modeling together with data analysis (Xu, S., S. Malinchik, Th. Kraft, B. Brenner, and L. C. Yu. 1997. X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle. Biophys. J. 73:000-000) indicate that in a relaxed muscle, cross-bridges are distributed in three populations: those that are ordered on the thick filament helix and those that are disordered; and within the disordered population, some cross-bridges are detached and some are weakly attached to actin. One critical conclusion of the present study is that the apparent order <--> disorder transition as a function of temperature is not due to an increase/decrease in thermal motion (temperature factor) for the entire population, but a redistribution of cross-bridges among the three populations. Changing the temperature leads to a change in the fraction of cross-bridges located on the helix, while changing the ionic strength at a given temperature affects the disordered population leading to a change in the relative fraction of cross-bridges detached from and weakly attached to actin. Since the redistribution is reversible, we suggest that there is an equilibrium among the three populations of cross-bridges.

X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle

X-ray diffraction patterns were obtained from skinned rabbit psoas muscle under relaxing and rigor conditions over a wide range of ionic strengths (50-170 mM) and temperatures (1 degree C-30 degrees C). For the first time, an intensification of the first actin-based layer line is observed in the relaxed muscle. The intensification, which increases with decreasing ionic strength at various temperatures, including 30 degrees C, parallels the formation of weakly attached cross-bridges in the relaxed muscle. However, the overall intensities of the actin-based layer lines are low. Furthermore, the level of diffuse scattering, presumably a measure of disorder among the cross-bridges, is little affected by changing ionic strength at a given temperature. The results suggest that the intensification of the first actin layer line is most likely due to the cross-bridges weakly bound to actin, and that the orientations of the weakly attached cross-bridges are hardly distinguishable from the detached cross-bridges. This suggests that the orientations of the weakly attached cross-bridges are not precisely defined with respect to the actin helix, i.e., nonstereospecific. Intensities of the myosin-based layer lines are only marginally affected by changing ionic strength, but markedly by temperature. The results could be explained if in a relaxed muscle the cross-bridges are distributed between a helically ordered and a disordered population with respect to myosin filament structure. Within the disordered population, some are weakly attached to actin and others are detached. The fraction of cross-bridges in the helically ordered assembly is primarily a function of temperature, while the distribution between the weakly attached and the detached within the disordered population is mainly affected by ionic strength. Some other notable features in the diffraction patterns include a approximately 1% decrease in the pitch of the myosin helix as the temperature is raised from 4 degrees C to 20 degrees C.

The Effect of Thin Filament Activation on the Attachment of Weak Binding Cross-Bridges: A Two-Dimensional X-Ray Diffraction Study on Single Muscle Fibers

To study possible structural changes in weak cross-bridge attachment to actin upon activation of the thin filament, two-dimensional (2D) x-ray diffraction patterns of skinned fibers from rabbit psoas muscle were recorded at low and high calcium concentration in the presence of saturating concentrations of MgATPgammaS, a nucleotide analog for weak binding states. We also studied 2D x-ray diffraction patterns recorded under relaxing conditions at an ionic strength above and below 50 mM, because it had been proposed from solution studies that reducing ionic strength below 50 mM also induces activation of the thin filament. For this project a novel preparation had to be established that allows recording of 2D x-ray diffraction patterns from single muscle fibers instead of natural fiber bundles. This was required to minimize substrate depletion or product accumulation within the fibers. When the calcium concentration was raised, the diffraction patterns recorded with MgATPgammaS revealed small changes in meridional reflections and layer line intensities that could be attributed in part to the effects of calcium binding to the thin filament (increase in I380, decrease in first actin layer line intensity, increase in I59) and in part to small structural changes of weakly attached cross-bridges (e.g., increase in I143 and I72). Calcium-induced small-scale structural rearrangements of cross-bridges weakly attached to actin in the presence of MgATPgammaS are consistent with our previous observation of reduced rate constants for attachment and detachment of cross-bridges with MgATPgammaS at high calcium. Yet, no evidence was found that weakly attached cross-bridges change their mode of attachment toward a stereospecific conformation when the actin filament is activated by adding calcium. Similarly, reducing ionic strength to less than 50 mM does not induce a transition from nonstereospecific to stereospecific attachment.

Response of Equatorial X-Ray Reflections and Stiffness to Altered Sarcomere Length and Myofilament Lattice Spacing in Relaxed Skinned Cardiac Muscle

Low angle x-ray diffraction measurements of myofilament lattice spacing (D(1,0)) and equatorial reflection intensity ratio (I(1,1)/I(1,0)) were made in relaxed skinned cardiac trabeculae from rats. We tested the hypothesis that the degree of weak cross-bridge (Xbr) binding, which has been shown to be obligatory for force generation in skeletal muscle, is modulated by changes in lattice spacing in skinned cardiac muscle. Altered weak Xbr binding was detected both by changes in I(1,1)/I(1,0) and by measurements of chord stiffness (chord K). Both measurements showed that, similar to skeletal muscle, the probability of weak Xbr binding at 170-mM ionic strength was significantly enhanced by lowering temperature to 5 degrees C. The effects of lattice spacing on weak Xbr binding were therefore determined under these conditions. Changes in D(1,0), I(1,1)/I(1,0), and chord K by osmotic compression with dextran T500 were determined at sarcomere lengths (SL) of 2.0 and 2.35 micro m. At each SL increasing [dextran] caused D(1,0) to decrease and both I(1,1)/I(1,0) and chord K to increase, indicating increased weak Xbr binding. The results suggest that in intact cardiac muscle increasing SL and decreasing lattice spacing could lead to increased force by increasing the probability of initial weak Xbr binding.

A Model of Cross-Bridge Attachment to Actin in the A·M·ATP State Based on X-Ray Diffraction from Permeabilized Rabbit Psoas Muscle

A model of cross-bridges binding to actin in the weak binding A*M*ATP state is presented. The modeling was based on the x-ray diffraction patterns from the relaxed skinned rabbit psoas muscle fibers where ATP hydrolysis was inhibited by N-phenylmaleimide treatment (S. Xu, J. Gu, G. Melvin, L. C. Yu. 2002. Biophys. J. 82:2111-2122). Calculations included both the myosin filaments and the actin filaments of the muscle cells, and the binding to actin was assumed to be single headed. To achieve a good fit, considerable flexibility in the orientation of the myosin head and the position of the S1-S2 junction is necessary, such that the myosin head can bind to a nearby actin whereas the tail end was kept in the proximity of the helical track of the myosin filament. Hence, the best-fit model shows that the head binds to actin in a wide range of orientations, and the tail end deviates substantially from its lattice position in the radial direction (approximately 60 A). Surprisingly, the best fit model reveals that the detached head, whose location thus far has remained undetected, seems to be located close to the surface of the myosin filament. Another significant requirement of the best-fit model is that the binding site on actin is near the N terminus of the actin subunit, a position distinct from the putative rigor-binding site. The results support the idea that the essential role played by the weak binding states M*ATP <--> A*M*ATP for force generation lies in its flexibility, because the probability of attachment is greatly increased, compared with the weak binding M*ADP*P(i) <--> A*M*ADP*P(i) states.

Stabilization of Helical Order in the Thick Filaments by Blebbistatin: Further Evidence of Coexisting Multiple Conformations of Myosin

The degree of helical order of the thick filament of mammalian skeletal muscle is highly dependent on temperature and the nature of the ligand. Previously, we showed that there was a close correlation between the conformation of the myosin heads on the surface of the thick filaments and the extent of their helical order. Helical order required the heads to be in the closed conformation. In addition, we showed that, with the same ligand bound at the active site, three conformations of myosin coexisted in equilibrium. Hitherto, however, there was no detectable helical order as measured by x-ray diffraction under the temperatures studied for myosin with MgADP and the nucleotide-free myosin, raising the possibility that the concept of multiple conformations has limited validity. In this study, blebbistatin was used to stabilize the closed conformation of myosin. The degree of helical order is substantially improved with MgATP at low temperature or with MgADP or in the absence of nucleotide. The thermodynamic parameters of the disorder<-->order transition and the characteristics of the ordered array were not significantly altered by binding blebbistatin. The simplest explanation is that the binding of blebbistatin increases the proportion of myosin in the closed conformation from being negligible to substantial. These results provide further evidence for the coexistence of multiple conformations of myosin under a wide range of conditions and for the closed conformation being directly coupled to helical order.

Characterizations of Cross-Bridges in the Presence of Saturating Concentrations of MgAMP-PNP in Rabbit Permeabilized Psoas Muscle

Several earlier studies have led to different conclusions about the complex of myosin with MgAMP-PNP. It has been suggested that subfragment 1 of myosin (S1)-MgAMP-PNP forms an S1-MgADP-like state, an intermediate between the myosin S1-MgATP and myosin S1-MgADP states or a mixture of cross-bridge states. We suggest that the different states observed result from the failure to saturate S1 with MgAMP-PNP. At saturating MgAMP-PNP, the interaction of myosin S1 with actin is very similar to that which occurs in the presence of MgATP. 1) At 1 degrees C and 170 mM ionic strength the equatorial x-ray diffraction intensity ratio I11/I10 decreased with an increasing MgAMP-PNP concentration and leveled off by approximately 20 mM MgAMP-PNP. The resulting ratio was the same for MgATP-relaxed fibers. 2) The two dimensional x-ray diffraction patterns from MgATP-relaxed and MgAMP-PNP-relaxed bundles are similar. 3) The affinity of S1-MgAMP-PNP for the actin-tropomyosin-troponin complex in solution in the absence of free calcium is comparable with that of S1-MgATP. 4) In the presence of calcium, I11/I10 decreased toward the relaxed value with increasing MgAMP-PNP, signifying that the affinity between cross-bridge and actin is weakened by MgAMP-PNP. 5) The degree to which the equatorial intensity ratio decreases as the ionic strength increases is similar in MgAMP-PNP and MgATP. Therefore, results from both fiber and solution studies suggest that MgAMP-PNP acts as a non hydrolyzable MgATP analogue for myosin.

Behavior of N-Phenylmaleimide-Reacted Muscle Fibers in Magnesium-Free Rigor Solution

Using x-ray diffraction and mechanical stiffness, the response of N-phenylmaleimide (NPM)-reacted cross-bridges to solutions containing different amounts of ATP and Mg2+ has been studied. In relaxing solution containing greater than millimolar amounts of ATP and Mg2+, NPM-treated muscle fibers give x-ray diffraction patterns and stiffness records, which are nearly indistinguishable from those of untreated relaxed fibers. In a solution devoid of added ATP, but with Mg2+ (rigor(+Mg) solution), the muscle fibers still give x-ray diffraction patterns and mechanical responses characteristic of relaxed muscle. The new finding reported here is that in a solution devoid of both ATP and Mg2+ (rigor(-Mg) solution containing EDTA with no added ATP), NPM-reacted cross-bridges do give rigor-like behavior. This is the first report that NPM-reacted cross-bridges, at least in the presence of EDTA, are capable of going into a strongly binding conformation.

Helical order in myosin filaments and evidence for one ligand inducing multiple myosin conformations.

The basic processes of muscle contraction are well understood: it is a result of cyclic interactions between myosin and actin, driven by the energy of ATP hydrolysis. Since the availability of the crystal structures of the contractile proteins, and with the advent of single molecule assays, the field has made great strides in understanding the underlying processes. However, the details of the mechanism of transduction of chemical to mechanical energy still remain largely unresolved. One of the obstacles is that most of the studies at the molecular level are based on isolated, in vitro systems, e.g. the atomic structure of the myosin head is known but not its complex with actin and EM reconstruction is based on isolated filaments. The link between the information obtained from the in vitro systems and the actual processes occurring in intact muscle is still largely missing. The aim of our efforts is to provide such a link.