Polarization from a Helix of Fluorophores and its Relation to that Obtained from Muscle

Expressions for the polarization of fluorescence from fluorophores in a helical array have been obtained. These predictions have been compared with experimental data obtained by reacting fluorescent S-1 subfragments of myosin (extrinsically labeled with N-(iodoacetylaminoethyl)-5-napthylamine-1-sulfonic acid [1,5-IAEDANS]) with glycerated rabbit psoas fibers in rigor. A comparison with data obtained by directly labeling fibers with this fluorophore is also presented.     

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°.     

Orientation of cross-bridges in skeletal muscle measured with a hydrophobic probe.

Cis-parinaric acid (PA) binds to a hydrophobic pocket formed between the heavy chain of myosin subfragment-1 (S1) and the 41-residue N-terminal of essential light chain 1 (A1). The binding is strong (Ka = 5.6 x 10(7) M-1) and rigid (polarization = 0.334). PA does not bind to myofibrils in which A1 has been extracted or replaced with alkali light chain 2 (A2). As in the case of S1 labeled with other probes, polarization of fluorescence of S1-PA added to myofibrils depended on fractional saturation of actin filament with S1, i.e., on whether the filaments were fully or partially saturated with myosin heads. Because fluorescence quantum yield of PA is enhanced manyfold upon binding, and because PA binds weakly to myofibrillar structures other then A1, the dye is a convenient probe of cross-bridge orientation in native muscle fibers. The polarization of a fiber irrigated with PA was equal to the polarization of S1-PA added to fibers at nonsaturating concentration. Cross-linking of S1 added to fibers at nonsaturating concentration showed that each S1 bound to two actin monomers of a thin filament. These results suggest that in rigor rabbit psoas muscle fiber each myosin cross-bridge binds to two actins.     

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.     

Polarized ultraviolet fluorescence microscopic study of the structural changes in muscle fiber contractile proteins. V. The possible nature of the conformational rearrangements in heavy meromyosin in fiber relaxation]

The mode and degree of tryptophanyl orientation relative to muscle fiber axes within hydrophobic and hydrophylic sites of myosin macromolecule in the presence of a fluorescence quencher (acrylamide, NO-3) during rigor and relaxation of glycerinated muscle fibers were studied using the polarized ultraviolet fluorescent microscopy. It was shown that myosin tryptophanyls both in LMM and HMM are oriented with their short axes along the longer axis of muscle fiber. Tryptophanyls in LMM have a more pronounced anisotropy of orientation in comparison with the fluorophore orientation anisotropy in hydrophobic sites of HMM. During the muscle fiber relaxation, conformational changes in HMM take place owing to which a section of polypeptide chain with a hydrophilic fluorophore is probably submerged deep into the macromolecule and becomes unapprochable to the quencher.     

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.     

Differential behavior of two cysteine residues on the myosin head in muscle fibers

We have previously shown that the orientation of (iodoacetamido)tetramethylrhodamine labels on SH1 thiol of S-1 moieties changes when MgADP is added to the fibers in rigor [Borejdo, J., Assulin, O., Ando, T., & Putnam, S. (1982) J. Mol. Biol. 158, 391-414. Burghardt, T.P., Ando, T., & Borejdo, J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7515-7519]. Here we report the results of experiments in which the SH2 of S-1 was labeled with maleimidorhodamine. The specificity of modification of thiols was checked by measuring the stoichiometry of attached dye, by determining the extent of the decrease in EDTA (K+)- and Ca2+-ATPase activities, and by the localization of the dyes on peptides containing SH1 and/or SH2. Labeled S-1 was diffused into single glycerinated fibers of rabbit psoas muscle, and the orientation of chromophores was measured by fluorescence detected dichroism. The dye attached to SH1 was oriented at 65 degrees with respect to the fiber axis in rigor and at 51 degrees in the presence of MgADP, regardless of whether SH2 was modified or not. The dye on SH2 was oriented near 42 degrees both in the presence and in the absence of ADP, regardless of whether SH1 was modified or not. Our results show that rhodamine oriented differently when attached to SH2 compared with when attached to SH1 and that in the former placement it was not sensitive to MgADP. We think this indicates that the SH2-containing region has a mobility different from that of the SH1-containing region, i.e., that this is evidence for internal flexibility of S-1.     

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.     

Fluorescence polarization of skeletal muscle fibers labeled with rhodamine isomers on the myosin heavy chain.

Fluorescence polarization was used to examine orientational changes of Rhodamine probes in single, skinned muscle fibers from rabbit psoas muscle following either photolysis of caged nucleotides or rapid length changes. Fibers were extensively and predominantly labeled at SH1 (Cys-707) of the myosin heavy chain with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine. Results from spectroscopic experiments utilizing the two Rhodamine isomers were quite similar. Following photolysis of either caged ATP or caged ADP, probes promptly reoriented toward the muscle fiber axis. Changes in the fluorescence polarization signals with transients elicited by the photolysis of caged ATP in the presence of saturating Ca2+ greatly preceded active force generation. Photolysis of caged ADP caused only a small, rapid decrease in force but elicited changes in the fluorescence polarization signals with time course and amplitude similar to those following photolysis of caged ATP. Fluorescence polarization signals were virtually unchanged by rapid length steps in both rigor and active muscle fibers. These results indicate that structural changes monitored by Rhodamine probes at SH1 are not associated directly with the force-generating event of muscle contraction. However, the fluorescence polarization transients were slightly faster than the estimated rate of cross-bridge detachment following photolysis of caged ATP, suggesting that the observed structural changes at SH1 may be involved in the communication pathway between the nucleotide- and actin-binding sites of myosin.     

Probing cross-bridge angular transitions using multiple extrinsic reporter groups.

15N- and 2H-substituted maleimido-TEMPO spin label ([15N,2H]MTSL) and the fluorescent label 1,5-IAEDANS were used to specifically modify sulfhydryl 1 of myosin to study the orientation of myosin cross-bridges in skeletal muscle fibers. The electron paramagnetic resonance (EPR) spectrum from muscle fibers decorated with labeled myosin subfragment 1 ([15N,2H]MTSL-S1) or the fluorescence polarization spectrum from fibers directly labeled with 1,5-IAEDANS was measured from fibers in various physiological conditions. The EPR spectra from fibers with the fiber axis oriented at 90 degrees to the Zeeman field show a clear spectral shift from the rigor spectrum when the myosin cross-bridge binds MgADP. This shift is attributable to a change in the torsion angle of the spin probe from cross-bridge rotation and is observable due mainly to the improved angular resolution of the substituted probe. The EPR data from [15N,2H]MTSL-S1 decorating fibers are combined with the fluorescence polarization data from the 1,5-IAEDANS-labeled fibers to map the global angular transition of the labeled cross-bridges due to nucleotide binding by an analytical method described in the accompanying paper [Burghardt, T. P., & Ajtai, K. (1992) Biochemistry (preceding paper in this issue)]. We find that the spin and fluorescent probes are quantitatively consistent in the finding that the actin-bound cross-bridge rotates through a large angle upon binding MgADP. We also find that, if the shape of the cross-bridge is described as an ellipsoid with two equivalent minor axes, then cross-bridge rotation takes place mainly about an axis parallel to the major axis of the ellipsoid. This type of rotation may imitate the rotation motion of cross-bridges during force generation.     

Steady-state fluorescence polarization studies of the orientation of myosin regulatory light chains in single skeletal muscle fibers using pure isomers of iodoacetamidotetramethylrhodamine.

The regulatory light chain (RLC) from chicken gizzard myosin was covalently modified on cysteine 108 with either the 5- or 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLCs were purified by fast protein liquid chromatography and characterized by reverse-phase high-performance liquid chromatography (HPLC), tryptic digestion, and electrospray mass spectrometry. Labeled RLCs were exchanged into the native myosin heads of single skinned fibers from rabbit psoas muscle, and the ATR dipole orientations were determined by fluorescence polarization. The 5- and 6-ATR dipoles had distinct orientations, and model orientational distributions suggest that they are more than 20 degrees apart in rigor. In the rigor-to-relaxed transition (sarcomere length 2.4 microm, 10 degrees C), the 5-ATR dipole became more perpendicular to the fiber axis, but the 6-ATR dipole became more parallel. This orientation change was absent at sarcomere length 4.0 microm, where overlap between myosin and actin filaments is abolished. When the temperature of relaxed fibers was raised to 30 degrees C, the 6-ATR dipoles became more parallel to the fiber axis and less ordered; when ionic strength was lowered from 160 mM to 20 mM (5 degrees C), the 6-ATR dipoles became more perpendicular to the fiber axis and more ordered. In active contraction (10 degrees C), the orientational distribution of the probe dipoles was similar but not identical to that in relaxation, and was not a linear combination of the orientational distributions in relaxation and rigor.     

Caldesmon weakens the bonding between myosin heads and actin in ghost fibers

Earlier studies using polarized microphotometry have shown that caldesmon inhibits the alterations in structure and flexibility of actin in ghost fibers that take place upon the binding of myosin heads (Ga?azkiewicz et al. (1987) Biochim. Biophys. Acta 916, 368-375). The present investigations, performed with an IAEDANS label attached to myosin subfragment 1 (S-1), revealed that this inhibition results from the weakening of the binding between myosin heads and actin as indicated by the caldesmon-induced increase in the random movement of S-1. Parallel experiments with actin labeled at Cys-374 demonstrated that this effect of caldesmon is transmitted to the C-terminus of the actin molecule resulting in a conformational adjustment in this region of the molecule.     

Fluorescent modification and orientation of myosin sulfhydryl 2 in skeletal muscle fibers

We describe a protocol for the selective covalent labeling of the sulfhydryl 2 (SH2) on the myosin cross-bridge in glycerinated muscle fibers using the sulfhydryl-selective label 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The protocol promotes the specificity of IANBD by using the ability to protect sulfhydryl 1 (SH1) from modification by binding the cross-bridge to the actin filament and using cross-bridge-bound MgADP to promote the accessibility of SH2. We determined the specificity of the probe using fluorescence gel scanning of fiber-extracted proteins to isolate the probe on myosin subfragment 1 (S1), limited proteolysis of the purified S1 to isolate the probe on the 20-kilodalton fragment of S1, and titration of the free SH1's on purified S1 using the radiolabeled SH1-specific reagent [14C]iodoacetamide or enzymatic activity measurements. We estimated the distribution of the IANBD on the fiber proteins to be approximately 77% on SH2, approximately 5% on SH1, and approximately 18% on troponin I. We characterized the angular distribution of the IANBD on cross-bridges in fibers when the fibers are in rigor, in relaxation, in the presence of MgADP, and in isometric contraction using wavelength-dependent fluorescence polarization [Ajtai, K., & Burghardt, T. P. (1987) Biochemistry 26, 4517-4523]. With wavelength-dependent fluorescence polarization we use the ability to rotate the transition dipole in the molecular frame using excitation wavelength variation to investigate the three angular degrees of freedom of the cross-bridge.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Orientation of the N-terminal lobe of the myosin regulatory light chain in skeletal muscle fibers

The orientation of the N-terminal lobe of the myosin regulatory light chain (RLC) in demembranated fibers of rabbit psoas muscle was determined by polarized fluorescence. The native RLC was replaced by a smooth muscle RLC with a bifunctional rhodamine probe attached to its A, B, C, or D helix. Fiber fluorescence data were interpreted using the crystal structure of the head domain of chicken skeletal myosin in the nucleotide-free state. The peak angle between the lever axis of the myosin head and the fiber or actin filament axis was 100—110° in relaxation, isometric contraction, and rigor. In each state the hook helix was at an angle of ～40° to the lever/filament plane. The in situ orientation of the RLC D and E helices, and by implication of its N- and C-lobes, was similar in smooth and skeletal RLC isoforms. The angle between these two RLC lobes in rigor fibers was different from that in the crystal structure. These results extend previous crystallographic evidence for bending between the two lobes of the RLC to actin-attached myosin heads in muscle fibers, and suggest that such bending may have functional significance in contraction and regulation of vertebrate striated muscle.     

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.     

Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy

To study the orientation and dynamics of myosin, we measured fluorescence polarization of single molecules and ensembles of myosin decorating actin filaments. Engineered chicken gizzard regulatory light chain (RLC), labeled with bisiodoacetamidorhodamine at cysteine residues 100 and 108 or 104 and 115, was exchanged for endogenous RLC in rabbit skeletal muscle HMM or S1. AEDANS-labeled actin, fully decorated with labeled myosin fragment or a ratio of approximately 1:1000 labeled:unlabeled myosin fragment, was adhered to a quartz slide. Eight polarized fluorescence intensities were combined with the actin orientation from the AEDANS fluorescence to determine the axial angle (relative to actin), the azimuthal angle (around actin), and RLC mobility on the <10 ms timescale. Order parameters of the orientation distributions from heavily labeled filaments agree well with comparable measurements in muscle fibers, verifying the technique. Experiments with HMM provide sufficient angular resolution to detect two orientations corresponding to the two heads in rigor. Experiments with S1 show a single orientation intermediate to the two seen for HMM. The angles measured for HMM are consistent with heads bound on adjacent actin monomers of a filament, under strain, similar to predictions based on ensemble measurements made on muscle fibers with electron microscopy and spectroscopic experiments.     

Is the SII portion of the cross-bridge in glycerinated rabbit psoas fibers compliant in the rigor state?

To see whether the SII portion of the cross-bridge in rigor fibers is longitudinally compliant, we chemically cross-linked with dimethyl suberimidate the entire rod portion (including the SII portion) of myosin onto the surface of thick filaments in glycerinated rabbit psoas fibers, and studied the effect of the SII fixation on the stiffness of the rigor fibers. The cross-linking of fiber segments with full filament overlap increased the rigor stiffness by approximately 25%. Almost the same absolute amount of the stiffness increase was also observed in rigor fibers with half- or no filament overlap after the cross-linking, and a similar but somewhat larger increment of stiffness was observed in fiber segments cross-linked in relaxing solution. These results indicate that the stiffness increase is not produced by the fixation of the SII portion onto the thick filament surface, but is caused instead by the cross-linking of some parallel elastic elements in muscle, and therefore indicate that the SII portion of the cross-bridge is hardly longitudinally compliant in rigor fibers.     

Observation of two orientations from rigor cross-bridges in glycerinated muscle fibers

The fluorescence polarization from rhodamine labels specifically attached to the fast-reacting thiol of the myosin cross-bridge in glycerinated muscle fibers has been measured to determine the angular distribution of the cross-bridges in different physiological states of the fibers as a function of temperature. To investigate the fibers at temperatures below 0 degree C, we have added glycerol to the bathing solution as an anti-freezing agent. We find that the fluorescence polarization from the rhodamine probe detects distinct angular distributions of the cross-bridges in isometric-active, rigor, MgADP, and low ionic strength relaxed fibers at 4 degrees C. We also find that the rigor cross-bridges in the presence of glycerol can maintain at least two distinct orientations relative to the actin filament, one dominant at temperatures T greater than 2 degrees C and another dominant at T less than -10 degrees C. MgADP cross-bridges in the presence of glycerol maintain approximately the same orientation for all temperatures investigated. The rigor cross-bridge orientation at T less than -10 degrees C is similar to both the MgADP cross-bridge orientation in the presence of glycerol and the active muscle cross-bridge orientation at 4 degrees C. These findings show that the rigor cross-bridge in the presence of glycerol has at least two distinct orientations while attached to actin: one of them dominant at high temperature, the other dominant at low temperature or when MgADP is present. The latter orientation resembles that present in isometric-active fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative studies on the polarization optical properties of striated muscle. I. Birefringence changes of rabbit psoas muscle in the transition from rigor to relaxed state

The changes in birefringence in the rigor to relax transition of single triton-extracted rabbit psoas muscle fibers have been investigated with quantitative polarized light techniques. The total birefringence of rest lenght fibers in rigor was (1.46 +/- 0.08) x 10(-3) and increased to (1.67 +/- 0.05) x 10(-3) after Mg-ATP relaxation. Pyrophosphate relaxation increased the total birefringence only slightly, whereas subsequent Mg-ATP relaxation elicited the maximum increase in birefringence. Changes in lattice spacing did not account for the total increase in birefrigence during relaxation. Moreover, the increase in total birefringence was attributable to increases in intrinsic birefringence as well as form birefringence. No change in birefringence was exhibited upon exposure to a relaxation solution after myosin extraction. Synthetic myosin filaments were prepared and treated with relaxation and rigor solutions. The negatively stained filaments treated with a rigor solution had gross irregular projections at either end, while the filaments treated with a relaxing solution were more spindle shaped. The results are compatible with the view that the subfragment-2 moieties of myosin angle away from the myosin aggregates (light meromyosin) to permit the attachment of the subfragment-1 moieties to actin.