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.     

Study of structural changes of contractile muscle proteins with the aid of polarization ultraviolet fluorescence microscopy. 1. Conformational changes of F-actin in the muscle fiber caused by ATP and its analogs]

Increase of anisotropy of F-actin fluorescence of balanus and rabbit muscle fibers under the influence of ATP, AMP and pyrophosphate in EGTA presence was detected by means of the polarized ultraviolet (UV) fluorescent microscopy methods. The fluorescence anisotropy changes are assumed to be associated with the conformational changes in the actin. ATP cause more noticeable changes of actin structure, than pyrophosphate and AMP. The conformational changes in the actin of balanus and rabbit muscle fibres were similar. ATP and its analogs induced also decrease of UV fluorescence anisotropy of A-band which appears to be associated with conformational changes in myosin. It was siggested that the changes in fluorescence of anisotropy of A-bands are due to structural changes in both HMM and LMM parts of myosin molecule.     

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.     

The effect of caldesmon and tropomyosin from smooth muscles on the motility of myosin head in ghost muscle fibers

The effects of caldesmon and smooth muscle tropomyosin on the motility of myosin subfragment I (SI) modified by N-(iodoacetyl)-N'-(1-naphtyl-5-sulfo)-ethylenediamine (1.5-IAEDANS) was studied in myosin-, troponin- and tropomyosin-free rabbit ghost muscle fibers using the polarized microphotometry technique. It was found that the fluorescence anisotropy initiated by the 1.5-IAEDANS-SI arrangement in the fibers is higher in the presence of tropomyosin than in its absence. Caldesmon diminishes the fluorescence anisotropy of the fibers. Data from a kinetic analysis suggest that the motility of fluorophores in the presence of tropomyosin in thin filaments is markedly decreased. Caldesmon weakens the effect of tropomyosin on the fluorescent label motility. It was supposed that caldesmon and tropomyosin initiate conformational changes in myosin heads which are accompanied by loosening or strengthening of their bonds with F-actin, respectively. Caldesmon inhibits the effect induced by tropomyosin.     

Effect of phosphorylation of light chain myosin and Ca2+ on the conformation of F-actin during skeletal muscle contraction

The dependence of polarized fluorescence of rhodaminylphalloin specifically bound to F-actin and the tension developed by a fiber upon phosphorylation of myosin (18.5 kD) light chains as well as on the concentration of free Ca2+ was observed during the contraction of glycerinated rabbit skeletal muscle fibers. Still greater changes in the polarized fluorescence and higher values of tension were recorded for fibers with phosphorylated light chains at low (0.6 microM) Ca2+ concentrations as well as for those with dephosphorylated light chains at high (10 microM) Ca2+ concentrations. It is concluded that phosphorylation of myosin light chains modulates skeletal muscle contraction. The mechanisms of modulation involve conformational changes in F-actin.     

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.     

The effect of Mg-ADP on the structural state of actin in the F-actin-myosin subfragment-1 complex]

Using polarized microfluorometry techniques, a study was made on the orientation and mobility of fluorescent probes 1,5-IAEDANS and rhomadin-phalloidin, located in various parts of actin, muscle fibers free of myosin, tropomyosin and troponin (ghost fibres) being used. It was found that the binding of a myosin subfragment 1 (S1) to actin induced changes in polarized fluorescence of the fibers. The analysis of these data showed that the formation of actin-S1 and actin-S1-ADP complexes in a muscle fiber resulted in a decrease in the angle between the thin filaments and the emission dipole of phalloidin-rhodamine, as well as in an increase of the mobility of this dye. In the experiments with the 1,5-IAEDANS label the angle of emission dipole increased, while the mobility of the label decreased. These changes were smaller in the presence of Mg-ADP than in its absence. It is assumed that the changes in actin monomer structure occur when a myosin head interacts with actin. These changes are expressed as those in orientation and mobility of large and small domains of actin in thin filaments. The domain orientation in actomyosin complex changes, influenced by Mg-ADP. The data obtained allow to propose the involvement of interdomain motions of some parts of actin monomer in the mechanisms of muscle contraction.     

Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle

Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7 degrees relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**.M**.ADP.Pi (weak binding) and A--.M--.ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the "closed" position caldesmon "freezes" the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.     

Changes in the structural state of myosin filaments in glycerol-treated fibers of rabbit skeletal muscles induced by phosphorylation of myosin light chains

Using the polarization microfluorimetry method, it was demonstrated that the increase in the degree of phosphorylation of myosin light chains (LC2) in extended single glycerinated fibers from rabbit psoas muscle changes the anisotropy of polarized fluorescence both tryptophan residue in the rod parts of the myosin molecule and the fluorescent label-N (iodoacetyl-aminoethyl)-5-naphthylamine-1-sulfonate (1,5-IAEDANS) bound to the SH1-group in myosin molecule heads. The changes in fluorescence anisotropy during LC2 phosphorylation were observed, when the measurements were performed only in the presence of 5 mM MgCl2. It was suggested that in the presence of MgCl2 the phosphorylation of LC2 associated with myosin heads changes their orientation and causes conformational shifts in the myosin filament core.     

Structural changes in contractile proteins of muscle fibers studied by polarization ultraviolet fluorescence microscopy. X. The effect of ATP, Ca2+, pH change and ionic strength of the washing solution on the structural state of thick filaments

By the method of polarized ultraviolet fluorescence microscopy, effects of ATP, Ca2+, changes in pH and ionic strength of washing solution of the structural state of thick filaments in both actin-free muscle fibers of rabbit and anisotropic discs (A-discs) of glycerinated fibers of crab were studied. The dependence of tryptophan fluorescence anisotropy of thin filaments upon physico-chemical parameters (compounds) of washing solution has been found. The structural state of thick filaments was suggested to be influenced by ATP, Ca2+, changes in pH and ionic strength of washing solution.     

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.     

Polarized fluorescence depletion reports orientation distribution and rotational dynamics of muscle cross-bridges

The method of polarized fluorescence depletion (PFD) has been applied to enhance the resolution of orientational distributions and dynamics obtained from fluorescence polarization (FP) experiments on ordered systems, particularly in muscle fibers. Previous FP data from single fluorescent probes were limited to the 2(nd)- and 4(th)-rank order parameters, and , of the probe angular distribution (beta) relative to the fiber axis and , a coefficient describing the extent of rapid probe motions. We applied intense 12-micros polarized photoselection pulses to transiently populate the triplet state of rhodamine probes and measured the polarization of the ground-state depletion using a weak interrogation beam. PFD provides dynamic information describing the extent of motions on the time scale between the fluorescence lifetime (e.g., 4 ns) and the duration of the photoselection pulse and it potentially supplies information about the probe angular distribution corresponding to order parameters above rank 4. Gizzard myosin regulatory light chain (RLC) was labeled with the 6-isomer of iodoacetamidotetramethylrhodamine and exchanged into rabbit psoas muscle fibers. In active contraction, dynamic motions of the RLC on the PFD time scale were intermediate between those observed in relaxation and rigor. The results indicate that previously observed disorder of the light chain region in contraction can be ascribed principally to dynamic motions on the microsecond time scale.     

State of the contractile apparatus in the development of a pathological process in muscle. IV. Effect of Ca2+ on the process of contraction nodule formation and on the ATPase activity of muscle actomyosin in Zenker's necrosis

Using phase-contrast and polarized ultraviolet (UV) fluorescent microscopy, the structure of single muscle fibres was studied in the course of the contraction module formation during Zenker's necrosis. The degree of manifestation of destructive changes in the contractile system was shown to depend upon the concentration of extracellular Ca-ions. With decreasing Ca2+ concentration, the fibre loses the ability to form contraction nodules peculiar to the Zenker necrosis, and the development of this process is interrupted at the stage of sarcomere supercontraction. The UV fluorescent anisotropy pattern of fibre regions, conforming with the contraction nodules, suggests the occurrence of a more pronounced disorganization of contractile system in the presence of Ca2+. The ATPase activity of actomyosin isolated from altered muscle was studied to appreciate the functional state of the contractile system. This actomyosin was found to be inactivated 1.5 times as much as that isolated from muscles treated during Zenker's necrosis in calcium-free media.     

Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers.

To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers.     

Interaction of isoforms of subfragment-1 of myosin, containing fluorescently labelled alkaline light chains, with muscle fiber actin

Using polarization microfluorimetry, the interaction of myosin subfragment 1 (S1) isoforms containing alkali light chains A1 and A2 respectively (S1(A1) and S1(A2] with F-actin of single glycerinated rabbit skeletal muscle fibers was studied. The alkali light chains of S1 were substituted by reassociation for A1 or A2 chains modified by a fluorescent label (1.5-IAEDANS) at the single SH-group located in the C-terminus. It was found that in S1(A1) bound to muscle fiber F-actin the mobility of the fluorescent label is lower than in S1(A2). At the same time the S1(A1) and S1(A2) interaction with F-actin induces similar changes in polarized fluorescence of rhodamine linked to falloidine which, in turn, is specifically bound to F-actin. It is concluded that the both S1 isoforms bind to F-actin and produce similar effects on the conformational state of actin filaments in muscle fibers. Local differences between S1(A1) and S1(A2) seem to be due to the interaction of the N-terminus of A1 within S1(A1) with the C-terminal region of actin.     

The effect of myosin light chain phosphorylation and Mg2+ on the conformation of myosin in thick filaments of glycerinated fibers of rabbit skeletal muscle

It has been shown by polarization microfluorimetry that phosphorylation of myosin light chain 2, in stretched single glycerinated fibers of rabbit skeletal muscle, results in changes in polarized fluorescence anisotropy of both the tryptophan residues of myosin molecules and the fluorescent label, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, associated with the fast-reacting thiol group in myosin heads. These changes are also dependent on the presence or absence of Mg2+ in the medium: they are most pronounced in the presence of 5 mM MgCl2. It is assumed that both Mg2+ binding to myosin and phosphorylation of light chain 2 associated with myosin heads induce structural changes in myosin filaments of muscle fibres which are expressed as changes in the orientation of myosin heads and in the conformation of myosin rods.     

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.     

Optical depolarization changes in single, skinned muscle fibers. Evidence for cross-bridge involvement.

Optical ellipsometry studies of single, skinned muscle fibers conducted on the diffraction orders have yielded spectra that are sensitive to the state of the fiber. The linearly polarized light field vector becomes elliptically polarized as it passes through the fiber and may be collected at the diffraction orders. Fibers that have been subjected to extraction of myosin (0.6 M KCl) retain a weak diffraction pattern and exhibit a substantially decreased depolarization of incident linearly polarized light. A significant decrease in polarization is seen in skinned fibers that are subject to an increase in pH from 7.0 to 8.0. This increase in pH results in a decrease of approximately 30% in the depolarization angle of single fibers. The major decrease in depolarization angle that we observe at pH 8.0 is consistent with the notion that as cross-bridges move out from the shaft of the thick filament, their ability to cause depolarization of the incident linearly polarized light decreases. This interpretation is also consistent with the work of Ueno and Harrington where the decrease in the ability to cross-link S-1 and S-2 to the thick filament at pH 8.2 suggests cross-bridge movement away from the thick filament. A large decrease in birefringence, seen after treatment of skinned fibers with alpha-chymotrypsin, appears to be related to the breakdown of myosin into rod, S-1, heavy meromyosin, and light meromyosin.     

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°.     

The disappearance of the dependence of actin-myosin interaction on the phosphorylation of myosin light chains in the "freezing" of the structure of heavy meromyosin by a bifunctional reagent

Using glycerinated muscle fibers, free of myosin, tropomyosin and troponin, a study was made of the structural state of F-actin modified by N-(iodoacetyl)-N'-(1-naphthyl-5-sulfo)-ethylendiamine (1.5-IAEDANS) and by rhodaminyl--phalloin at decoration of thin filaments with a proteolytic fragment of myosin--heavy meromyosin containing phosphorylated and dephosphorylated myosin light chains. The heavy meromyosin used has three SH-groups of heavy chain SH1, SH2 and SH chi modified by bifunctional reagent N,N'-n-phenylmaleimide (SH1-SH2, SH2-SH chi). At decoration of thin filaments with heavy meromyosin, some changes in polarized fluorescence of rhodaminyl--phalloin and 1.5-IAEDANS independent of phosphorylation of myosin light chains were found. Fluorescence anisotropy of the fiber was found to depend primarily on the character of heavy chain of SH-group modification. The ability of heavy chains to change their conformations is supposed to play an important role in the mechanism of myosin system modulation of muscle contraction.