Refined structure of bony fish muscle myosin filaments from low-angle X-ray diffraction data

Application of X-ray diffraction methods to the elucidation of the arrangement of the myosin heads on myosin filaments in resting muscles is made simpler when the muscles themselves are well ordered in 3D. Bony fish muscle for the vertebrates and insect flight muscle for the invertebrates are the muscles of choice for this analysis. The rich, well-sampled, low-angle X-ray diffraction pattern from bony fish muscle has previously been modelled with an R-factor of 3.4% between observed and calculated transforms on the assumption that the two heads in one myosin molecule have the same shape. However, recent evidence from other kinds of analysis of other muscles has shown that this assumption may not be valid. There is evidence that the motor domain of one head in each pair may interact with the neck region of the second head. This possibility has been tested directly in the present analysis which extends the X-ray modelling of fish muscle myosin filaments by permitting independent shape changes of the two heads in one molecule. The new model, with a computed R-factor of 1.19% against 56 independent reflections, shows that in fish muscle also there is a marked asymmetry in the organisation of each head pair.     

Structure and nucleotide-dependent changes of thick filaments in relaxed and rigor plaice fin muscle

The myosin crossbridge array, positions of non-crossbridge densities on the backbone, and the A-band "end filaments" have been compared in chemically skinned, unfixed, uncryoprotected relaxed, and rigor plaice fin muscles using the freeze-fracture, deep-etch, rotary-shadowing technique. The images provide a direct demonstration of the helical packing of the myosin heads in situ in relaxed muscle and show rearrangements of the myosin heads, and possibly of other myosin filament proteins, when the heads lose ATP on going into rigor. In the H-zone these changes are consistent with crossbridge changes previously shown by others using freeze-substitution. In addition, new evidence is presented of protein rearrangements in the M-region (bare zone), associated with the transition from the relaxed to the rigor state, including a 27-nm increase in the apparent width of the M-region. This is interpreted as being mostly due to loss or rearrangement of a nonmyosin (M9) protein component at the M-region edge. The structure and titin periodicity of the end-filaments are described, as are suggestions of titin structure on the myosin filament backbone.     

X-ray diffraction testing for weak-binding crossbridges in relaxed bony fish muscle fibres at low ionic strength

Equatorial X-ray diffraction patterns from single skinned fibres from bony fish muscle (turbot) were obtained with the fibres at 6 degrees C bathed in relaxing solutions of 170 down to 26 mM ionic strength. Diffraction patterns from rigor fibres were also obtained as controls. Unlike fibres from rabbit muscle, which show very clear evidence of substantial crossbridge formation at low ionic strength in what is mechanically a rapid equilibrium ("weak-binding") state (Brenner et al., 1982), diffraction patterns from bony fish fibres showed only a small change in relative peak intensities at low ionic strength (26 mM) compared with normal (170 mM) ionic strength. However, there was a slight ordering of the filament lattice at low ionic strength. The specimen temperature used (about 6 degrees C) was not far from the normal physiological temperature of the fish. Likewise, only a small change was seen by Xu et al. (1987) in patterns from frog fibres at low ionic strength at 2 to 6 degrees C. (Rabbit fibres previously studied, where large changes were seen at temperatures of 5 to 20 degrees C, were about 17 to 32 degrees C below physiological.) The I11/I10 ratio for fish fibres at 26 mM ionic strength was actually lower than that for rabbit even at normal ionic strength. This may be associated with an intrinsic structural difference between these muscles or alternatively with the disordering of the crossbridge helix in rabbit muscle found at low temperature by Wray (1987), and could support the view that rabbit fibres at 5 degrees C and normal ionic strength may already have a significant population of weak-binding crossbridges.     

The myosin cross-bridge cycle and its control by twitchin phosphorylation in catch muscle

The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATPgammaS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca(2+)] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.     

Structural role of tropomyosin in muscle regulation: analysis of the x-ray diffraction patterns from relaxed and contracting muscles

Previous work has shown that there are significant differences in the X-ray diffraction patterns obtained from relaxed and contracting muscles. We show that some of these changes can be explained in terms of a small movement (~ 5 to 15 Å) of the tropomyosin molecules in the groove of the actin helix. The position of the tropomyosin in relaxed skeletal muscle is such that it might physically block or at least structurally alter the cross-bridge attachment site on actin, whereas in contracting skeletal muscle the tropomyosin moves to a position well clear of the attachment site. The movement of the tropomyosin molecules is apparently smaller in molluscan muscles during tonic contraction than in vertebrate skeletal muscle. We suggest a possible relationship between the smaller movement of the tropomyosin and the “catch” response of molluscan muscles.We also show that any increase of intensity on the 59 Å and 51 Å layer-lines is most likely to be associated with some extra mass (HMM S-1) attaching to the actin molecules. Such a change cannot be explained in terms of a change in tropomyosin structure or in the order within the thin filaments. Since changes on these two layer-lines have been observed during contraction, this provides good evidence for cross-bridge attachment to actin in contracting muscles.     

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.     

Cardiac myosin binding protein C and its phosphorylation regulate multiple steps in the cross-bridge cycle of muscle contraction

Cardiac myosin binding protein C (c-MyBPC) is a thick filament protein that is expressed in cardiac sarcomeres and is known to interact with myosin and actin. While both structural and regulatory roles have been proposed for c-MyBPC, its true function is unclear; however, phosphorylation has been shown to be important. In this study, we investigate the effect of c-MyBPC and its phosphorylation on two key steps of the cross-bridge cycle using fast reaction kinetics. We show that unphosphorylated c-MyBPC complexed with myosin in 1:1 and 3:1 myosin:c-MyBPC stoichiometries regulates the binding of myosin to actin (K(D)) cooperatively (Hill coefficient, h) (K(D) = 16.44 ± 0.33 μM, and h = 9.24 ± 1.34; K(D) = 11.48 ± 0.75 μM, and h = 3.54 ± 0.67) and significantly decelerates the ATP-induced dissociation of myosin from actin (K(1)k(+2) values of 0.12 ± 0.01 and 0.22 ± 0.01 M(-1) s(-1), respectively, compared with a value of 0.42 ± 0.01 M(-1) s(-1) for myosin alone). Phosphorylation of c-MyBPC abolished the regulation of the association phase (K(1)k(+2) values of 0.32 ± 0.02 and 0.33 ± 0.01 M(-1) s(-1) at 1:1 and 3:1 myosin:c-MyBPC ratios, respectively) and also accelerated the dissociation of myosin from actin (K(1)k(+2) values of 0.23 ± 0.01 and 0.29 ± 0.01 M(-1) s(-1) at a 1:1 and 3:1 myosin:c-MyBPC ratios, respectively) relative to the dissociation of myosin from actin in the presence of unphosphorylated c-MyBPC. These results indicate a direct effect of c-MyBPC on cross-bridge kinetics that is independent of the thin filament that together with its phosphorylation provides a mechanism for fine-tuning cross-bridge behavior to match the contractile requirements of the heart.     

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.     

Interparticle effects in low-angle x-ray and neutron diffraction from chromatin.

Published diffraction data are critically reviewed, and replotted in a new way to show the variation with concentration of the 8- to 25- nm diffraction maximum. Most of the early data are found to be consistent with a single model for a liquid-type array of mutually repulsive particles, whose molecular weight is calculated to be that of a nucleosome or possibly a dimer. The data for all but the highest concentrations, where distortion due to dehydration is possible, support no particular model for the higher-order coiling of chains of nucleosomes, and cannot be used to support models for "native" chromatin. Only in the presence of excess salts or after isolation with polyamines is there aggregation in solution of nucleosomes, which then give peaks at 11 and 5.5 nm that do not change much with concentration. Recent work by the authors confirms that under some conditions nucleosome undergo a transition to a state whose diffraction is consistent with hexagonal packing of extended DNA to which histones are still attached. This state is probably responsible for much of the strong 2.7-nm peak previously obtained from certain samples, which was in some cases assigned to nucleosome structure. Only the peak at 3.7 nm is clearly attributable to the form factor of the isolated native nucleosome.     

X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle.

We have used a small angle scattering system assembled on the high flux multipole wiggler beam line at CHESS (Cornell) to make very accurate spacing measurements of certain meridional and layer-line reflections from contracting muscles. During isometric contraction, the actin 27.3 A reflection increases in spacing from its resting value by approximately 0.3%, and other actin reflections, including the 59 and 51 A off-meridional reflections, show corresponding changes in spacing. When tension is augmented or diminished by applying moderate speed length changes to a contracting muscle, changes in spacing in the range of 0.19-0.24% (when scaled to full isometric tension) can be seen. The larger difference between the resting and isometric spacings suggests either nonlinearity at low tension levels or the presence of a component related to activation itself. Myosin filaments also show similar increases in axial period during slow stretch, in addition to the well known larger change associated with activation. An actin spacing change of 0.25-0.3% can also be measured during a 2 ms time frame immediately after a quick release, showing that the elastic behavior is rapid. These observations of filament extensions totaling 2-3 nm per half-sarcomere may necessitate some significant revision of the interpretation of a number of mechanical experiments in muscle, in which it has usually been assumed that virtually all of the elasticity resides in the cross-bridges.     

Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle

The Ca2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83:3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC2) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (less than 1 ms) released to a shorter length and then reextended by approximately 200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 - e-kt) to obtain the value of ktr. In control fibers, ktr increased sigmoidally with increases in [Ca2+]; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 +/- 1.2 s-1 at 15 degrees C) than in rabbit psoas fibers (18.7 +/- 1.0 s-1). Phosphorylation of LC2 was accomplished by repeated Ca2+ activations (pCa 4.5) of the fibers in solutions containing 6 microM calmodulin and 0.5 microM myosin light chain kinase, a protocol that resulted in an increase in LC2 phosphorylation from approximately 10% in the control fibers to greater than 80% after treatment. After phosphorylation, ktr was unchanged at maximum or very low levels of Ca2+ activation. However, at intermediate levels of Ca2+ activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC2 phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology. 86:585-600), the effect of LC2 phosphorylation to increase the Ca2+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Distribution of mass in relaxed frog skeletal muscle and its redistribution upon activation.

Five orders of equatorial reflection were recorded from both relaxed and fully activated intact frog sartorius muscle using synchrotron x-ray radiation. Electron density maps of the myofilament lattice in axial projection were calculated from the integrated intensities by Fourier synthesis, using all possible phase combinations. These maps were evaluated systematically in terms of their compatibility with electron microscopically and biochemically derived properties of the lattice structure and with the minimum wavelength principle. For the relaxed state, one phase combination emerged as most consistent with these constraints: it shows a thick filament with a compact core surrounded by an annular shell of density. The distribution of mass suggests that the S-2 moiety of the myosin molecule is an integral part of the thick-filament backbone and the S-1 moiety makes up the shell and is tilted or slewed around the backbone. For the active state, there are two feasible maps, which differ according to whether or not the activation process is associated with phase inversion in two of the reflections. Both maps represent patterns of redistribution of mass upon activation in which the thick-filament backbone is practically unaffected and there is movement of density from the annular shell towards the thin filaments. In addition to this outward radial flux of density from the thick-filament periphery, the pattern of net mass transfer involves a pronounced azimuthal component in both cases. The total net mass transfer is equivalent to approximately 20% (no phase change) or approximately 40% (with phase change) of the S-1 mass. From the observed systematic increase in peak widths of the higher orders, the size of the crystalline domain in the myofilament lattice in the relaxed sartorius is estimated to be greater than 650 nm and the variations in myofilament lattice spacing among different myofibrils to be about +/- 3%. Furthermore, in the activated state, the equilibrium positions of the myofilaments are no longer well ordered, but are distributed statistically about the lattice points with a standard deviation of approximately 3 nm.     

Purification and properties of myosin from the "hatching muscle" (m. complexus) of geese

The present work is concerned with the study of myosin fractions prepared from the hatching muscle (m. complexus) and a control muscle (m. pectoralis) of the developing goose embryo. The m. complexus attained its maximum mass at hatching and in the 4-day-old bird the mass of this muscle was only one fourth of that recorded at hatching. The m. complexus was hypertrophied already on the 21st day. At days 21, 27 and 28 of incubation and at posthatching days myosin preparations were made from both muscles. Partial purification of myosins from both sources yielded a high molecular weight fraction characteristic of the adult bird and one other protein fraction with molecular mass half of myosin. Both preparations exhibited the characteristic properties of myosin. The lower molecular weight fraction was also shown to develop filamentous aggregates as did the higher molecular-weight, gel filtrated myosin. The phosphate content of the half molecular mass myosin fraction prepared from the embryonic m. complexus at days prior to hatching was considerably higher than that of the high molecular weight fraction and the predominant component was P-Arg. Since the embryonic myosin was still not available in the m. complexus of the 4-day-old birds and the hypertrophied muscle underwent regression after hatching it appears that this myosin fraction is actively involved in breaking through the shell during the hatching period in geese.     

The majority of potassium ions in muscle cells is adsorbed on beta- and gamma-carboxyl groups of myosin: potassium-ion-adsorbing carboxyl groups on myosin heads engage in cross-bridge formation during contraction.

High-molecular weight poly(ethylene glycol) (PEG-8000) in the bathing medium prolongs the survival of 2-mm-wide frog muscle segments with open ends. In a PEG-8000-containing medium Rb+, K+, and Na+ in the muscle segments reached new diffusion equilibrium in 2-4 hours. At this new equilibrium, the cell's preference of K+ over Na+ was preserved but very much weakened. Studies of the influence of pH on the equilibrium distribution of labelled Na+ in 2-mm-wide muscle segments confirmed the prediction that beta- and gamma-carboxyl groups, carried respectively on aspartic and glutamic acid residues of intracellular proteins, adsorb K+, Na+ and other monovalent cations. These carboxyl groups have a characteristic pKa between 3.65 and 4.25. A pKa of 3.85 was observed. These findings, when seen in the light of other relevant information available, led to the conclusion that beta- and gamma-carboxyl groups on myosin molecules adsorb--in a close contact one-ion-one-site fashion--the majority (67% to 80%) of K+ in resting muscle cells. Other evidence suggests that in muscle contraction, the K(+)-adsorbing beta- and gamma-carboxyl groups on myosin heads form salt linkages with cationic sites on actin, displacing and releasing the adsorbed K+. Present and earlier findings together offer support for an earlier suggestion that the formation and dissociation of these salt-linkages may underlie the force-generating, cyclic formation and dissociation of cross-bridges during muscle contraction.     

Time-resolved changes in equatorial x-ray diffraction and stiffness during rise of tetanic tension in intact length-clamped single muscle fibers.

We report the first time-resolved x-ray diffraction studies on tetanized intact single muscle fibers of the frog. The 10, 11, 20, 21, 30, and Z equatorial reflections were clearly resolved in the relaxed fiber. The preparation readily withstood 100 1-s duration (0.4-s beam exposure) tetani at 4 degrees C (less than 4% decline of force and no deterioration in the 10, 11 equatorial intensity ratio at rest or during activation). Equatorial intensity changes (10 and 11) and fiber stiffness led tension (t1/2 lead 20 ms at 4 degrees C) during the tetanus rise and lagged during the isometric phase of relaxation. These findings support the existence of a low force cross-bridge state during the rise of tetanic tension and isometric relaxation that is not evident at the tetanus plateau. In "fixed end" tetani lattice expansion occurred with a time course similar to stiffness during the tetanus rise. During relaxation, lattice spacing increased slightly, while the sarcomere length remained isometric, but underwent large changes after the "shoulder" of tension. Under length clamp control, lattice expansion during the tetanus rise was reduced or abolished, and compression (2%) of the lattice was observed. A lattice compression is predicted by certain cross-bridge models of force generation (Schoenberg, M. 1980. Biophys. J. 30:51-68; Schoenberg, M. 1980. Biophys. J. 30:69-78).     

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) have been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated Mr of 229 X 10(3), and can be divided into a long alpha-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two "hinges" for the 6 S/10 S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg2+ ATP is bound to a pocket between two opposing alpha/beta domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.     

Fine structure in near-field and far-field laser diffraction patterns from skeletal muscle fibers.

Regions of muscle fibers that are many sarcomeres in length and uniform with regard to striation spacing, curvature, and tilt have been observed by light microscopy. We have investigated the possibility that these sarcomere domains can explain the fine structure in optical diffraction patterns of skeletal muscle fibers. We studied near-field and far-field diffraction patterns with respect to fiber translation and to masking of the laser beam. The position of diffracted light in the near-field pattern depends on sarcomere length and position of the diffracting regions within the laser beam. When a muscle fiber was translated longitudinally through a fixed laser beam, the fine structural lines in the near-field diffraction pattern moved in the same direction and by the same amount as the fiber movement. Translation of the muscle fiber did not result in fine structure movement in the far-field pattern. As the laser beam was incrementally masked from one side, some fine structural lines in both the near-field and far-field diffraction patterns changed in intensity while others remained the same. Eventually, all the fine structural lines broadened and decreased in intensity. Often a fine structural line increased in intensity or a dark area in the diffraction pattern became brighter as the laser beam was restricted. From these results we conclude that the fine structure in the laser diffraction pattern is due to localized and relatively uniform regions of sarcomeres (domains) and to cross interference among light rays scattered by different domains.