EPR evidence for nucleotide effects on attached cross-bridges.

We have used electron paramagnetic resonance (EPR) to study the effects of ATP and nucleotide analogs (mainly AMPPNP) on the orientation (measured by conventional EPR) and microsecond rotational dynamics (measured by saturation transfer EPR, STEPR) of spin-labeled myosin heads, both in glycerinated muscle fibers and in solutions of purified S1 and actin. Attachment to actin was determined by stiffness measurements in fibers and by covalent cross-linking in acto-S1. Our goal is to determine whether these nucleotides induce conformations of attached cross-bridges that have head orientations or motions significantly different from rigor. While all heads are immobile and similarly oriented in fibers in rigor, relaxation by ATP produces great orientational disorder that is dynamic on the microsecond time scale. AMPPNP produces intermediate amounts of both disorder and motion. However, even at saturating levels of AMPPNP, there are two principal resolved populations of heads, whose motion and orientation are indistinguishable from those of rigor and relaxation. Thus, there is no evidence that AMPPNP induces a myosin head conformation differing in either orientation or rotational motion from the rigor and relaxed states. Since fiber stiffness is not significantly decreased by AMPPNP, even though up to 50% of myosin heads are dynamically disordered, some of the dynamically disordered heads are probably in attached cross-bridges. Simultaneous measurements of binding and orientation of labeled S1 diffused into fibers or cross-linked to actin, indicate that AMPPNP-bound heads are dynamically disordered only when detached from actin. Thus, AMPPNP detaches one of the two heads of each cross-bridge without decreasing the cross-bridge's stiffness. In contrast to AMPPNP, ATP does induce considerable microsecond rotational mobility within cross-linked acto-S1, indicating that dynamic disorder of myosin heads may occur during the attached phase of an active cross-bridge cycle. Thus we have identified two nucleotide-induced cross-bridge conformations that are rotationally different from rigor, and similar conformations may play a role in the force-generation process.

Centrosomal components immunologically related to tektins from ciliary and flagellar microtubules.

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific- or cross-specific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidissima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48-50 kDa in isolated spindles and centrosomes from CHO cells.

Myosin heads have a broad orientational distribution during isometric muscle contraction: time-resolved EPR studies using caged ATP.

To study the orientation of spin-labeled myosin heads in the first few seconds after the production of saturating ATP, we have used a laser flash to photolyze caged ATP during EPR data acquisition. Rabbit psoas muscle fibers were labeled with maleimide spin label, modifying 60% of the myosin heads without impairing muscle fiber biochemical and physiological activity (ATPase and force). The muscle bundles were incubated for 30 min with 5 mM caged ATP prior to the UV flash. The flash, from an excimer laser, liberated 2-3 mM ATP, generating maximum force in the presence of Ca2+ and relaxing fully in the absence of Ca2+. Control experiments, using fibers decorated with labeled myosin subfragment, showed that the flash liberates sufficient ATP to saturate myosin active sites in all regions of the muscle bundles. To increase the time resolution, and to minimize the time of the contraction, we followed in time the intensity at a single spectral position (P2), which is associated with the high degree of orientational order in rigor. ATP liberation produced a rapid decrease of P2 with liberation of ATP, indicating a large decrease in orientational order in both relaxation and contraction. This transient was absent when caged AMP was used, ruling out nonspecific effects of the UV flash and subsequent photochemistry. The steady-state level of P2 during contraction was almost as low as that reached in relaxation, although the duration of the steady state was much more brief in contraction. Upon depletion of ATP in contraction, the P2 intensity reverted to the original rigor level, accompanied by development of rigor tension. The steady-state results obtained in the brief contractions induced by caged ATP are quantitatively consistent with those obtained in longer contractions by continuously perfusing fibers with ATP. In isometric contraction, most (88% +/- 4%) of the heads are in a population characterized by a high degree of axial disorder, comparable to that observed for all heads in relaxation. Since the stiffness of these fibers in contraction is 80% of the stiffness in rigor, it is likely that most of the heads in this highly disoriented population are attached to actin in contraction and that most actin-attached heads in contraction are in this disoriented population.

Orientational disorder and motion of weakly attached cross-bridges.

In a relaxed muscle fiber at low ionic strength, the cross-bridges may well be in states comparable to the one that precedes the cross-bridge power stroke (Schoenberg, M. 1988. Adv. Exp. Med. Biol. 226:189-202). Using electron paramagnetic resonance (EPR) and (saturation transfer) electron paramagnetic resonance (ST-EPR) techniques on fibers labeled with maleimide spin label, under low ionic strength conditions designed to produce a majority of weakly-attached heads, we have established that (a) relaxed labeled fibers show a speed dependence of chord stiffness identical to that of unlabeled, relaxed fibers, suggesting similar rapid dissociation and reassociation of cross-bridges; (b) the attached relaxed heads at low ionic strength are nearly as disordered as in relaxation at physiological ionic strength where most of the heads are detached from actin; and (c) the microsecond rotational mobility of the relaxed heads was only slightly restricted compared to normal ionic strength, implying great motional freedom despite attachment. The differences in head mobility between low and normal ionic strength scale with filament overlap and are thus due to acto-myosin interactions. The spectra can be modeled in terms of two populations: one identical to relaxed heads at normal ionic strength (83%), the other representing a more oriented population of heads (17%). The spectrum of the latter is centered at approximately the same angle as the spectrum in rigor but exhibits larger (40 degrees) axial probe disorder with respect to the fiber axis. Alternatively, assuming that the chord stiffness is proportional to the fraction of attached crossbridges, the attached fraction must be even more disordered than 400, with rotational mobility nearly as great as for detached cross-bridges.

Effects of AMPPNP on the orientation and rotational dynamics of spin-labeled muscle cross-bridges.

We have used electron paramagnetic resonance (EPR) to investigate the orientation, rotational motion, and actin-binding properties of rabbit psoas muscle cross-bridges in the presence of the nonhydrolyzable nucleotide analogue, 5'-adenylylimido-diphosphate (AMPPNP). This analogue is known to decrease muscle tension without affecting its stiffness, suggesting an attached cross-bridge state different from rigor. We spin-labeled the SH1 groups on myosin heads and performed conventional EPR to obtain high-resolution information about the orientational distribution, and saturation transfer EPR to measure microsecond rotational motion. At 4 degrees C and 100 mM ionic strength, we find that AMPPNP increases both the orientational disorder and the microsecond rotational motion of myosin heads. However, computer analysis of digitized spectra shows that no new population of probes is observed that does not match either rigor or relaxation in both orientation and motion. At 4 degrees C, under nearly saturating conditions of 16 mM AMPPNP (Kd = 3.0 mM, determined from competition between AMPPNP and an ADP spin label), 47.5 +/- 2.5% of myosin heads are dynamically disoriented (as in relaxation) without a significant decrease in rigor stiffness, whereas the remainder are rigidly oriented as in rigor. The oriented heads correspond to actin-attached heads in a ternary complex, and the disoriented heads correspond to detached heads, as indicated by EPR experiments with spin-labeled subfragment 1 (S1) that provide independent measurements of orientation and binding. We take these findings as evidence for a single-headed cross-bridge that is as stiff as the double-headed rigor cross-bridge. The data are consistent with a model in which, in the presence of saturating AMPPNP, one head of each cross-bridge binds actin about 10 times more weakly, whereas the remaining head binds at least 10 times more strongly, than extrinsic S1. Thus, although there is no evidence for heads being attached at nonrigor angles, the attached cross-bridge differs from that of rigor. The heterogeneous behavior of heads is probably due to steric effects of the filament lattice.

Effect of ADP on the orientation of spin-labeled myosin heads in muscle fibers: a high-resolution study with deuterated spin labels.

We have used electron paramagnetic resonance (EPR) to determine the effects of ADP on the orientational distribution of nitroxide spin labels attached to myosin heads in skinned rabbit psoas muscle fibers. To maximize the specificity of labeling, we spin-labeled isolated myosin heads (subfragment 1) on a single reactive thiol (SH1) and diffused them into unlabeled muscle fibers. To maximize spectral and orientational resolution, we used perdeuterated spin labels, 2H-MSL and 2H-IASL, eliminating superhyperfine broadening and thus narrowing the line widths. Two different spin labels were used, with different orientation relative to the myosin head, to ensure that the results are not affected by unfavorable probe orientation. In rigor, a very narrow three-line spectrum was observed for both spin labels, indicating a narrow orientational distribution, as reported previously (Thomas & Cooke, 1980). ADP induced very slight changes in the spectrum, corresponding to very slight (but significant) changes in the orientational distribution. These changes were quantified by a digital analysis of the spectra, using a two-step simplex fitting procedure (Fajer et al., 1990). First, the magnetic tensor values and line widths were determined by fitting the spectrum of a randomly oriented sample. Then the spectrum of oriented fibers was fit to a model by assuming a Gaussian distribution of the tilt angle (theta) and twist angle (phi) of the nitroxide principal axes relative to the fiber axis. A single-Gaussian distribution resulted in inadequate fits, but a two-component model gave excellent results. ADP induces a small (less than 5 degrees) rotation of the major components for both spin labels, along with a similarly small increase of disorder about the average positions.

A novel electron paramagnetic resonance spin label and its application to study the cross-bridge cycle.

We have used a novel alpha-iodoketone spin-label (IKSL) to study myosin head orientation and cross-bridge dynamics in the putative pre-powerstroke state. Possible perturbation of the cross-bridge cycle by the label was assayed by the sinusoidal analysis method (Kawai and Brandt, 1980; Kawai and Zhao, 1993), which determines the rate constants of the elementary steps in the cycle. A comparison of the rates obtained from unlabeled and IKSL fibers revealed small (10-20%) changes in the ATP hydrolysis rate and in the rate constants of the elementary steps. The labeling induced small changes (< 13%) in the distribution of the cross-bridges among the various intermediate states. Pre-powerstroke cross-bridges were induced by aluminum fluoride in the presence of Ca2+ and ATP. In this state, force development is inhibited, but a large proportion (40%) of the cross-bridges are still attached to the thin filament. We have used conventional electron paramagnetic resonance to measure orientation, and have found that the pre-powerstroke heads are as disordered as in relaxation. Their mobility, as determined by saturation transfer electron paramagnetic resonance, was significantly restricted. Assuming that stiffness is proportional to the fraction of strongly attached heads, the rotational correlation time of the bound heads is estimated to be tau r = approximately 150-400 microseconds.