Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers.

Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys108 with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys108-5 or Cys108-6) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys108-6 dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to length changes are consistent with a partition of approximately 90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted approximately 30 degrees for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys108-6 dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca2+. Polarization ratios for Cys108-6 in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys108-6 dipole were restricted to a cone of approximately 20 degrees half-angle, and those of Cys108-5 dipole to a cone of approximately 25 degrees half-angle. These values changed little between relaxation, active contraction, and rigor. Cys108-5 showed very small-amplitude tilting toward the fiber axis for both stretches and releases in active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that the RLC undergoes a large angle change (approximately 60 degrees) between these two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about its own axis.     

Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle.

The orientation of the light-chain region of myosin heads in relaxed, rigor, and isometrically contracting fibers from rabbit psoas muscle was studied by fluorescence polarization. Cysteine 108 of chicken gizzard myosin regulatory light chain (cgRLC) was covalently modified with iodoacetamidotetramethylrhodamine (iodo-ATR). Native RLC of single glycerinated muscle fibers was exchanged for labeled cgRLC in a low [Mg2+] rigor solution at 30 degrees C. Troponin and troponin C removed in this procedure were replaced. RLC exchange had little effect on active force production. X-ray diffraction showed normal structure in rigor after RLC exchange, but loss of axial and helical order in relaxation. In isolated myofibrils labeled cgRLC was confined to the regions of the sarcomere containing myosin heads. The ATR dipoles showed a preference for orientations perpendicular to the fiber axis, combined with limited nanosecond rotational motion, in all conditions studied. The perpendicular orientation preference was more marked in rigor than in either relaxation or active contraction. Stretching relaxed fibers to sarcomere length 4 microns to eliminate overlap between actin- and myosin-containing filaments had little effect on the orientation preference. There was no change in orientation preference when fibers were put into rigor at sarcomere length 4.0 microns. Qualitatively similar results were obtained with ATR-labeled rabbit skeletal RLC.     

A birefringence study of changes in myosin orientation during relaxation of skinned muscle fibers induced by photolytic ATP release.

The birefringence of isolated skinned fibers from rabbit psoas muscle was measured continuously during relaxation from rigor produced by photolysis of caged ATP at sarcomere length 2.8-2.9 microns, ionic strength 0.1 M, 15 degrees C. Birefringence, the difference in refractive index between light components polarized parallel and perpendicular to the fiber axis, depends on the average degree of alignment of the myosin head domain with the fiber axis. After ATP release birefringence increased by 5.8 +/- 0.7% (mean +/- SE, n = 6) with two temporal components. A small fast component had an amplitude of 0.9 +/- 0.2% and rate constant of 63 s-1. By the completion of this component, the instantaneous stiffness had decreased to about half the rigor value, and the force response to a step stretch showed a rapid (approximately 1000 s-1) recovery phase. Subsequently a large slow birefringence component with rate constant 5.1 s-1 accompanied isometric force relaxation. Inorganic phosphate (10 mM) did not affect the fast birefringence component but accelerated the slow component and force relaxation. The fast birefringence component was probably caused by formation of myosin.ATP or myosin.ADP.Pi states that are weakly bound to actin. The average myosin head orientation at the end of this component is slightly more parallel to the fiber axis than in rigor.     

Orientation of spin-labeled light chain-2 exchanged onto myosin cross-bridges in glycerinated muscle fibers.

Electron paramagnetic resonance (EPR) spectroscopy has been used to study the angular distribution of a spin label attached to rabbit skeletal muscle myosin light chain 2. A cysteine reactive spin label, 3-(5-fluoro-2,4-dinitroanilino)-2,2,5,5- tetramethyl-1-pyrrolidinyloxy (FDNA-SL) was bound to purified LC2. The labeled LC2 was exchanged into glycerinated muscle fibers and into myosin and its subfragments. Analysis of the spectra of labeled fibers in rigor showed that the probe was oriented with respect to the fiber axis, but that it was also undergoing restricted rotations. The motion of the probe could be modeled assuming rapid rotational diffusion (rotational correlation time faster than 5 ns) within a "cone" whose full width was 70 degrees. Very different spectra of rigor fibers were obtained with the fiber oriented parallel and perpendicular to the magnetic field, showing that the centroid of each cone had the same orientation for all myosin heads, making an angle of approximately 74 degrees to the fiber axis. Binding of light chains or labeled myosin subfragment-1 to ion exchange heads immobilized the probes, showing that most of the motion of the probe arose from protein mobility and not from mobility of the probe relative to the protein. Relaxed labeled fibers produced EPR spectra with a highly disordered angular distribution, consistent with myosin heads being detached from the thin filament and undergoing large angular motions. Addition of pyrophosphate, ADP, or an ATP analogue (AMPPNP), in low ionic strength buffer where these ligands do not dissociate cross-bridges from actin, failed to perturb the rigor spectrum. Applying static strains as high as 0.16 N/mm2 to the labeled rigor fibers also failed to change the orientation of the spin label. Labeled light chain was exchanged into myosin subfragment-1 (S1) and the labeled S1 was diffused into fibers. EPR spectra of these fibers had a component similar to that seen in the spectra of fibers into which labeled LC2 had been exchanged directly. However, the fraction of disordered probes was greater than seen in fibers. In summary, the above data indicate that the region of the myosin head proximal to the thick filament is ordered in rigor, and disordered in relaxation.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin.

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.     

Polarization of fluorescence from single skinned glycerinated rabbit psoas fibers in rigor and relaxation

Single skinned glycerinated muscle fibers were labelled with the fluorescent dye N-(iodoacetylamino)-1-naphthylamine-5-sulfonic acid (1,5-IAEDANS). The heavy chain of myosin (EC 3.6.1.3) was labelled predominantly when the reaction was carried out in relaxation at 0 °C. Mechanical properties of skinned fibers were little affected by labelling with the fluorophore. Rigor tension developed upon transferring native or labelled skinned fibers from relaxing to rigor solutions lacking Ca²⁺ was very small but could be enhanced by progressively increasing Ca²⁺ concentration; the rigor tension decreased with increasing sarcomere length.Polarization of fluorescence of skinned fibers reacted with 1,5-IAEDANS was measured along the line of excitation as well as at 90° to it. The mean values of parallel and perpendicular components of polarization of labelled fibers measured at 0° were close to the values obtained for native fibers irrigated with 1,5-IAEDANS-labelled heavy meromyosin, fiber “ghosts” irrigated with labelled heavy meromyosin, and oriented bundles of myofibrils reacted with the same fluorophore. Skinned fibers stretched above the rest length and then irrigated with 1,5-IAEDANS-labelled heavy meromyosin gave rise to polarized fluorescence close to the values theoretically predicted for an assembly of helically arranged fluorophores. Using 90° detection system a satisfactory fit to the theory could be obtained from single fibers labelled with 1,5-IAEDANS and measured in rigor. The angle between the fiber axis and the direction of the emission dipole of 1,5-IAEDANS attached to subfragment-1 was estimated to be near 40°.     

Polarization of Tryptophan Fluorescence from Single Striated Muscle Fibers : A molecular probe of contractile state

Instrumentation has been developed to detect rapidly the polarization of tryptophan fluorescence from single muscle fibers in rigor, relaxation, and contraction. The polarization parameter (P) obtained by exiciting the muscle tryptophans with light polarized perpendicular to the long axis of the muscle fiber had a magnitude P (relaxation) > P (contraction) > P (rigor) for the three types of muscle fibers examined (glycerinated rabbit psoas, glycerinated dorsal longitudinal flight muscle of Lethocerus americanus, and live semitendinosus of Rana pipiens). P from single psoas fibers in rigor was found to increase as the sarcomere length increased but in relaxed fibers P was independent of sarcomere length. After rigor, pyrophosphate produced little or no change in P, but following an adenosine triphosphate (ATP)-containing solution, pyrophosphate produced a value of P that fell between the contraction and relaxation values. Sinusoidal or square wave oscillations of the muscle of amplitude 0.5–2.0% of the sarcomere length and frequency 1, 2, or 5 Hz were applied in rigor when the myosin cross-bridges are considered to be firmly attached to the thin filaments. No significant changes in P were observed in either rigor or relaxation. The preceding results together with our present knowledge of tryptophan distribution in the contractile proteins has led us to the conclusion that the parameter P is a probe of the contractile state of myosin which is probably sensitive to the orientation of the myosin S1 subfragment.     

Orientation of spin-labeled myosin heads in glycerinated muscle fibers.

We have used electron paramagnetic resonance (EPR) spectra to study spin labels selectively and rigidly attached to myosin heads in glycerinated rabbit psoas muscle fibers. Because the angle between the magnetic field and the principal axis of the probe determines the position of the EPR absorption line, spectra from labeled fibers oriented parallel to the magnetic field yielded directly the distribution of spin label orientations relative to the fiber axis. Two spin labels, having reactivities resembling iodoacetamide (IASL) and maleimide (MSL), were used. In rigor fibers with complete filament overlap, both labels displayed a narrow angular distribution, full width at half maximum approximately 15 degrees, centered at angles of 68 degrees (IASL) and 82 degrees (MSL). Myosin subfragments (heavy meromyosin and subfragment-1) were labeled and allowed to diffuse into fibers. The resulting spectra showed the same sharp angular distribution that was found for the labeled fibers. Thus is appears that virtually all myosin heads in a rigor fiber have the same orientation relative to the fiber axis, and this orientation is determined by the actomyosin bond. Experiments with stretched fibers indicated that the spin labels on the fraction of heads not interacting with actin filaments had a broad angular distribution. Addition of ATP to unstretched fibers under relaxing conditions produced orientational disorder, resulting in a spectrum almost indistinguishable from that of an isotropic distribution of probes. Addition of either an ATP analog (AMPPNP) or pyrophosphate produced partial disorder. That is a fraction of the probes remained sharply oriented as in rigor while a second fraction was in a disordered distribution similar to that of relaxed fibers.     

GFP-tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber

Myosin is the molecular motor in muscle-binding actin and executing a power stroke by rotating its lever arm through an angle of approximately 70 degrees to translate actin against resistive force. A green fluorescent protein (GFP)-tagged human cardiac myosin regulatory light chain (HCRLC) was constructed to study in situ lever arm orientation one molecule at a time by polarized fluorescence emitted from the GFP probe. The recombinant protein physically and functionally replaced the native RLC on myosin lever arms in the thick filaments of permeabilized skeletal muscle fibers. Detecting single molecules in fibers where myosin concentration reaches 300 microM is accomplished using total internal reflection fluorescence microscopy. With total internal reflection fluorescence, evanescent field excitation, supercritical angle fluorescence detection, and CCD detector pixel size limits detection volume to just a few attoliters. Data analysis manages both the perturbing effect of the TIR interface on probe emission and the effect of high numerical aperture collection of light. The natural myosin concentration gradient in a muscle fiber allows observation of fluorescence polarization from C-term GFP-tagged HCRLC exchanged myosin from regions in the thick filament containing low and high myosin concentrations. In rigor, cross-bridges at low concentration at the end of the thick filament maintain GFP dipole moments at two distinct polar angles relative to the fiber symmetry axis. The lower angle, where the dipole is nearly parallel to fiber axis, is more highly populated than the alternative, larger angle. Cross-bridges at higher concentration in the center of the thick filament are oriented in a homogeneous band at approximately 45 degrees to the fiber axis. The data suggests molecular crowding impacts myosin conformation, implying mutual interactions between cross-bridges alter how the muscle generates force. The GFP-tagged RLC is a novel probe to assess single-lever-arm orientation characteristics in situ.     

Saturation transfer electron paramagnetic resonance of spin-labeled muscle fibers. Dependence of myosin head rotational motion on sarcomere length

We have investigated the orientation and rotational mobility of spin-labeled myosin heads in muscle fibers as a function of the sarcomere length in the absence of ATP. An iodoacetamide spin label was used to label selectively two-thirds of the sulfhydryl-1 groups in glycerinated rabbit psoas muscle. Conventional electron paramagnetic resonance experiments were used to determine the orientation distribution of the probes relative to the fiber axis, and saturation transfer experiments were used to detect sub-millisecond rotational motion. When fibers are at sarcomere length 2.3 microns (full overlap), spin-labeled heads have a high degree of orientational order. The probes are in a single, narrow orientation distribution (full width 15 degrees), and they exhibit no detectable sub-millisecond rotational motion. When fibers are stretched (sarcomere length increased), either before or after labeling, disorder and microsecond mobility increase greatly, in proportion to the fraction of myosin heads that are no longer in the overlap zone between the thick and thin filaments. Saturation transfer difference spectra show that a fraction of myosin heads equal to the fraction outside the overlap zone have much more rotational mobility than those in fibers at full overlap, and almost as much as in synthetic myosin filaments. The most likely interpretation is that some of the probes, corresponding approximately to the fraction of heads in the overlap zone, remain oriented and immobile, while the rest are highly disordered (angular spread greater than 90 degrees) and mobile (microsecond rotational motion). Thus, it appears that myosin heads are rigidly immobilized by actin, but they rotate through large angles on the microsecond time-scale when detached from actin, even in the absence of ATP.     

Orientational dynamics of indane dione spin-labeled myosin heads in relaxed and contracting skeletal muscle fibers.

We have used electron paramagnetic resonance (EPR) spectroscopy to study the orientation and rotational motions of spin-labeled myosin heads during steady-state relaxation and contraction of skinned rabbit psoas muscle fibers. Using an indane-dione spin label, we obtained EPR spectra corresponding specifically to probes attached to Cys 707 (SH1) on the catalytic domain of myosin heads. The probe is rigidly immobilized, so that it reports the global rotation of the myosin head, and the probe's principal axis is aligned almost parallel with the fiber axis in rigor, making it directly sensitive to axial rotation of the head. Numerical simulations of EPR spectra showed that the labeled heads are highly oriented in rigor, but in relaxation they have at least 90 degrees (Gaussian full width) of axial disorder, centered at an angle approximately equal to that in rigor. Spectra obtained in isometric contraction are fit quite well by assuming that 79 +/- 2% of the myosin heads are disordered as in relaxation, whereas the remaining 21 +/- 2% have the same orientation as in rigor. Computer-simulated spectra confirm that there is no significant population (> 5%) of heads having a distinct orientation substantially different (> 10 degrees) from that in rigor, and even the large disordered population of heads has a mean orientation that is similar to that in rigor. Because this spin label reports axial head rotations directly, these results suggest strongly that the catalytic domain of myosin does not undergo a transition between two distinct axial orientations during force generation. Saturation transfer EPR shows that the rotational disorder is dynamic on the microsecond time scale in both relaxation and contraction. These results are consistent with models of contraction involving 1) a transition from a dynamically disordered preforce state to an ordered (rigorlike) force-generating state and/or 2) domain movements within the myosin head that do not change the axial orientation of the SH1-containing catalytic domain relative to actin.     

Bifunctional rhodamine probes of Myosin regulatory light chain orientation in relaxed skeletal muscle fibers

The orientation of the regulatory light chain (RLC) region of the myosin heads in relaxed skinned fibers from rabbit psoas muscle was investigated by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteine residues introduced into the RLC. Pure 1:1 BR-RLC complexes were exchanged into single muscle fibers in EDTA rigor solution for 30 min at 30 degrees C; approximately 60% of the native RLC was removed and stoichiometrically replaced by BR-RLC, and >85% of the BR-RLC was located in the sarcomeric A-bands. The second- and fourth-rank order parameters of the orientation distributions of BR dipoles linking RLC cysteine pairs 100-108, 100-113, 108-113, and 104-115 were calculated from polarized fluorescence intensities, and used to determine the smoothest RLC orientation distribution-the maximum entropy distribution-consistent with the polarized fluorescence data. Maximum entropy distributions in relaxed muscle were relatively broad. At the peak of the distribution, the "lever" axis, linking Cys707 and Lys843 of the myosin heavy chain, was at 70-80 degrees to the fiber axis, and the "hook" helix (Pro830-Lys843) was almost coplanar with the fiber and lever axes. The temperature and ionic strength of the relaxing solution had small but reproducible effects on the orientation of the RLC region.     

Model-independent analysis of the orientation of fluorescent probes with restricted mobility in muscle fibers

The orientation of proteins in ordered biological samples can be investigated using steady-state polarized fluorescence from probes conjugated to the protein. A general limitation of this approach is that the probes typically exhibit rapid orientational motion ("wobble") with respect to the protein backbone. Here we present a method for characterizing the extent of this wobble and for removing its effects from the available information about the static orientational distribution of the probes. The analysis depends on four assumptions: 1) the probe wobble is fast compared with the nanosecond time scale of its excited-state decay; 2) the orientational distributions of the absorption and emission transition dipole moments are cylindrically symmetrical about a common axis c fixed in the protein; 3) protein motions are negligible during the excited-state decay; 4) the distribution of c is cylindrically symmetrical about the director of the experimental sample. In a muscle fiber, the director is the fiber axis, F. All of the information on the orientational order of the probe that is available from measurements of linearly polarized fluorescence is contained in five independent polarized fluorescence intensities measured with excitation and emission polarizers parallel or perpendicular to F and with the propagation axis of the detected fluorescence parallel or perpendicular to that of the excitation. The analysis then yields the average second-rank and fourth-rank order parameters ( and ) of the angular distribution of c relative to F, and and , the average second-rank order parameters of the angular distribution for wobble of the absorption and emission transition dipole moments relative to c. The method can also be applied to other cylindrically ordered systems such as oriented lipid bilayer membranes and to processes slower than fluorescence that may be observed using longer-lived optically excited states.     

Effect of paratropomyosin on the increase in sarcomere length of rigor-shortened skeletal muscles

Paratropomyosin is a myofibrillar protein believed to weaken rigor linkages formed between actin and myosin. Using glycerinated fibers of rabbit psoas muscles, we studied the effect of paratropomyosin on the weakening of rigor linkages, which was followed in terms of the increase in sarcomere length of rigor-shortened muscles. The rigor tension developed was reduced to about 65% of the initial value within 10 min after the addition of purified paratropomyosin, whereas it remained constant for at least 3.5 h in control fibers. Paratropomyosin showed a stronger effect on the increase in sarcomere length of passively stretched fibers, which developed weaker rigor-tensions. The purpose of our research was to establish a rigor solution which would best permit the observation of the workings of paratropomyosin. The most successful rigor solution contained 0.2-0.25 M KCl, pH 5.5, at 5-10 degrees C. Under these conditions, the sarcomere length was easily increased from 2.4 to 3.6 micron, if rigor-contracted fibers were passively stretched after the addition of purified paratropomyosin. Because the experimental conditions coincide well with those of postmortem muscles, it is very probable that paratropomyosin plays an important role in restoration of the sarcomere length of rigor-shortened muscles, resulting in tenderization of meat during postrigor ageing.     

Motion of myosin cross-bridges in skeletal muscle fibers studied by time-resolved fluorescence anisotropy decay

The time-resolved fluorescence polarization anisotropy signal has been measured from fluorescent-labeled myosin cross-bridges in single glycerinated muscle fibers in the relaxed and rigor states. In one experimental configuration, the polarization of the excitation light and the fiber axis are aligned, and the anisotropy is sensitive to rotational motions of the probes about axes other than the fiber axis. The rotational correlation times are approximately 1000 ns for relaxed fibers and greater than 7000 ns for rigor fibers. In another experimental configuration, the excitation light polarization is perpendicular to the fiber axis, and its propagation vector has a component parallel to the fiber axis so that the anisotropy is sensitive to probe rotational motion about different axes, including the fiber axis. In this configuration, the rotational correlation times are approximately 300 ns for both relaxed and rigor fibers. The theory of rotational diffusion in a potential described in a related paper [Burghardt, T.P. (1985) Biophys. J. (in press)] is applied to the relaxed fiber data.     

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.     

Polarized fluorescence from epsilon-ADP incorporated into F-actin in a myosin-free single fiber: conformation of F-actin and changes induced in it by heavy meromyosin.

Fluorescent ADP analog, ε-ADP (1:N⁶-ethenoadenosine 5′-diphosphate), was incorporated into F-actin in a myosin-free ghost single fiber and polarized fluorescence measurements were performed under a microspectrophotometer to investigate the conformation of F-actin and the changes induced in it by heavy meromyosin and subfragment-1. Four components of polarized fluorescence were obtained by exciting the fiber with light polarized parallel and perpendicular to the long axis of the fiber and measuring the intensity of emission polarized parallel and perpendicular. From these data it was shown that F-actin in the fiber was not rigid but flexible, with a value for the elastic modulus for bending of 5.3 × 10^?17 dyn cm². The angles of absorption dipole and emission dipole of bound ε-ADP with the long axis of F-actin were both about 75 °.The binding of heavy meromyosin decreased the elastic modulus of F-actin by 30% and the angles of absorption and emission dipoles by 2.5 ° and 1.5 °, respectively. The molar ratios of bound heavy meromyosin and subfragment-1 to actin in the ghost fiber at saturation were 0.3 and 0.6, respectively, being smaller than those in solution.     

Myosin regulatory domain orientation in skeletal muscle fibers: application of novel electron paramagnetic resonance spectral decomposition and molecular modeling methods

Reorientation of the regulatory domain of the myosin head is a feature of all current models of force generation in muscle. We have determined the orientation of the myosin regulatory light chain (RLC) using a spin-label bound rigidly and stereospecifically to the single Cys-154 of a mutant skeletal isoform. Labeled RLC was reconstituted into skeletal muscle fibers using a modified method that results in near-stoichiometric levels of RLC and fully functional muscle. Complex electron paramagnetic resonance spectra obtained in rigor necessitated the development of a novel decomposition technique. The strength of this method is that no specific model for a complex orientational distribution was presumed. The global analysis of a series of spectra, from fibers tilted with respect to the magnetic field, revealed two populations: one well-ordered (+/-15 degrees ) with the spin-label z axis parallel to actin, and a second population with a large distribution (+/-60 degrees ). A lack of order in relaxed or nonoverlap fibers demonstrated that regulatory domain ordering was defined by interaction with actin rather than the thick filament surface. No order was observed in the regulatory domain during isometric contraction, consistent with the substantial reorientation that occurs during force generation. For the first time, spin-label orientation has been interpreted in terms of the orientation of a labeled domain. A Monte Carlo conformational search technique was used to determine the orientation of the spin-label with respect to the protein. This in turn allows determination of the absolute orientation of the regulatory domain with respect to the actin axis. The comparison with the electron microscopy reconstructions verified the accuracy of the method; the electron paramagnetic resonance determined that axial orientation was within 10 degrees of the electron microscopy model.     

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.