Myosin crossbridge orientation in demembranated muscle fibres studied by birefringence and X-ray diffraction measurements

Muscle contraction is generally thought to involve changes in the orientation of myosin crossbridges during their ATP-driven cyclical interaction with actin. We have investigated crossbridge orientation in equilibrium states of the crossbridge cycle in demembranated fibres of frog and rabbit muscle, using a novel combination of techniques: birefringence and X-ray diffraction. Muscle birefringence is sensitive to both crossbridge orientation and the transverse spacing of the contractile filament lattice. The latter was determined from the equatorial X-ray diffraction pattern, allowing accurate characterization of the orientation component of birefringence changes. We found that this component decreased when relaxed muscle fibres were put into rigor at rest length, and when either the ionic strength or temperature of relaxed fibres was lowered. In each case the birefringence decrease was accompanied by an increase in the intensity of the (1,1) equatorial X-ray reflection relative to that of the (1,0) reflection. When fibres that had been stretched largely to eliminate overlap between actin- and myosin-containing filaments were put into rigor, there was no change in the orientation component of the birefringence. When isolated myosin subfragment-1 was bound to these rigor fibres, the orientation component of the birefringence increased. The birefringence changes at rest length are likely to be due to changes in the orientation of myosin crossbridges, and in particular of the globular head region of the myosin molecules. In relaxed fibres from rabbit muscle, at 100 mM ionic strength, 15 degrees C, the long axis of the heads appears to be relatively well aligned with the filament axis. When fibres are put into rigor, or the temperature or ionic strength is lowered, the degree of alignment decreases and there is a transfer of crossbridge mass towards the actin-containing filaments.     

Characterization of the myosin adenosine triphosphate (M.ATP) crossbridge in rabbit and frog skeletal muscle fibers.

In the presence of ATP and absence of Ca2+, muscle crossbridges have either MgATP or MgADP.Pi bound at the active site (S. B. Marston and R. T. Tregear, Nature [Lond.], 235:22:1972). The behavior of these myosin adenosine triphosphate (M.ATP) crossbridges, both in relaxed skinned rabbit psoas and frog semitendinosus fibers, was analyzed. At very low ionic strength, T = 5 degrees C, mu = 20 mM, these crossbridges spend a large fraction of the time attached to actin. In rabbit, the attachment rate constants at low salt are 10(4) - 10(5) s-1, and the detachment rate constants are approximately 10(4) s-1. When ionic strength is increased up to physiological values by addition of 140 mM potassium propionate, the major effect is a weakening of the crossbridge binding constant approximately 30-40-fold. This effect occurs because of a large decrease, approximately 100-fold, in the crossbridge attachment rate constants. The detachment rate constants decrease only 2-3-fold. The effect of ionic strength on crossbridge binding in the fiber is very similar to the effect of ionic strength on the binding of myosin subfragment-1 to unregulated actin in solution. Thus, the effect of increasing ionic strength in fibers appears to be a direct effect on crossbridge binding rather than an effect on troponin-tropomyosin. The finding that crossbridges with ATP bound at the active site can and do attach to actin over a wide range of ionic strengths strongly suggests that troponin-tropomyosin keeps a muscle relaxed by blocking a step subsequent to crossbridge attachment. Thus, rather than troponin-tropomyosin serving to keep a muscle relaxed by inhibiting attachment, it seems quite possible that the main way in which troponin-tropomyosin regulates muscle activity is by preventing the weakly-binding relaxed crossbridges from going on through the crossbridge cycle into more strongly-binding states.     

X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle.

X-ray diffraction patterns were obtained from skinned rabbit psoas muscle under relaxing and rigor conditions over a wide range of ionic strengths (50-170 mM) and temperatures (1 degree C-30 degrees C). For the first time, an intensification of the first actin-based layer line is observed in the relaxed muscle. The intensification, which increases with decreasing ionic strength at various temperatures, including 30 degrees C, parallels the formation of weakly attached cross-bridges in the relaxed muscle. However, the overall intensities of the actin-based layer lines are low. Furthermore, the level of diffuse scattering, presumably a measure of disorder among the cross-bridges, is little affected by changing ionic strength at a given temperature. The results suggest that the intensification of the first actin layer line is most likely due to the cross-bridges weakly bound to actin, and that the orientations of the weakly attached cross-bridges are hardly distinguishable from the detached cross-bridges. This suggests that the orientations of the weakly attached cross-bridges are not precisely defined with respect to the actin helix, i.e., nonstereospecific. Intensities of the myosin-based layer lines are only marginally affected by changing ionic strength, but markedly by temperature. The results could be explained if in a relaxed muscle the cross-bridges are distributed between a helically ordered and a disordered population with respect to myosin filament structure. Within the disordered population, some are weakly attached to actin and others are detached. The fraction of cross-bridges in the helically ordered assembly is primarily a function of temperature, while the distribution between the weakly attached and the detached within the disordered population is mainly affected by ionic strength. Some other notable features in the diffraction patterns include a approximately 1% decrease in the pitch of the myosin helix as the temperature is raised from 4 degrees C to 20 degrees C.     

The effect of pyrophosphate on the rigor fibers of rabbit m. psoas fibers

The effect of MgPPi on the rigor force of glycerinated fibres in the range of ionic strength 75-250 mM at two temperatures 18 degrees and 5 degrees C was studied. At 18 degrees C the maximum of this effect was above the range of average ionic strength. At 5 degrees C the greatest effect of MgPPi was observed at low ionic strength.     

Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength

The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca2+ has been studied. When the ionic strength of an activating solution (containing Mg2+-ATP and Ca2+) was decreased by varying the KCl concentration from 120 to 5 mM at 20 degrees C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6 degrees C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca2+, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20 degrees C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca2+ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6 degrees C or at physiological ionic strength.     

Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium

Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from both the transition of crossbridges from a low- to high-force producing state as well as a transient detachment of crossbridges. From the temperature studies and dynamic stiffness measurements, the slow tension recovery phase most likely reflects the overall rate of crossbridge cycling. From the tension transient studies, it appears that crossbridges cycle slower and have a longer duty cycle in smooth muscle. In low [Ca2+]o at 20 degrees C, little effect was observed on the form or time course of the tension transients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Parallel inhibition of active force and relaxed fiber stiffness by caldesmon fragments at physiological ionic strength and temperature conditions: additional evidence that weak cross-bridge binding to actin is an essential intermediate for force generation.

Previously we showed that stiffness of relaxed fibers and active force generated in single skinned fibers of rabbit psoas muscle are inhibited in parallel by actin-binding fragments of caldesmon, an actin-associated protein of smooth muscle, under conditions in which a large fraction of cross-bridges is weakly attached to actin (ionic strength of 50 mM and temperature of 5 degrees C). These results suggested that weak cross-bridge attachment to actin is essential for force generation. The present study provides evidence that this is also true for physiological ionic strength (170 mM) at temperatures up to 30 degrees C, suggesting that weak cross-bridge binding to actin is generally required for force generation. In addition, we show that the inhibition of active force is not a result of changes in cross-bridge cycling kinetics but apparently results from selective inhibition of weak cross-bridge binding to actin. Together with our previous biochemical, mechanical, and structural studies, these findings support the proposal that weak cross-bridge attachment to actin is an essential intermediate on the path to force generation and are consistent with the concept that isometric force mainly results from an increase in strain of the attached cross-bridge as a result of a structural change associated with the transition from a weakly bound to a strongly bound actomyosin complex. This mechanism is different from the processes responsible for quick tension recovery that were proposed by Huxley and Simmons (Proposed mechanism of force generation in striated muscle. Nature. 233:533-538.) to represent the elementary mechanism of force generation.     

Interplay between passive tension and strong and weak binding cross- bridges in insect indirect flight muscle. A functional dissection by gelsolin-mediated thin filament removal

The interplay between passive and active mechanical properties of indirect flight muscle of the waterbug (Lethocerus) was investigated. A functional dissection of the relative contribution of cross-bridges, actin filaments, and C filaments to tension and stiffness of passive, activated, and rigor fibers was carried out by comparing mechanical properties at different ionic strengths of sarcomeres with and without thin filaments. Selective thin filament removal was accomplished by treatment with the actin-severving protein gelsolin. Thin filament, removal had no effect on passive tension, indicating that the C filament and the actin filament are mechanically independent and that passive tension is developed by the C filament in response to sarcomere stretch. Passive tension increased steeply with sarcomere length until an elastic limit was reached at only 6-7% sarcomere extension, which corresponds to an extension of 350% of the C filament. The passive tension-length relation of insect flight muscle was analyzed using a segmental extension model of passive tension development (Wang, K, R. McCarter, J. Wright, B. Jennate, and R Ramirez-Mitchell. 1991. Proc. Natl. Acad. Sci. USA. 88:7101-7109). Thin filament removal greatly depressed high frequency passive stiffness (2.2 kHz) and eliminated the ionic strength sensitivity of passive stiffness. It is likely that the passive stiffness component that is removed by gelsolin is derived from weak-binding cross-bridges, while the component that remains is derived from the C filament. Our results indicate that a significant number of weak-binding cross-bridges exist in passive insect muscle at room temperature and at an ionic strength of 195 mM. Analysis of rigor muscle indicated that while rigor tension is entirely actin based, rigor stiffness contains a component that resists gelsolin treatment and is therefore likely to be C filament based. Active tension and active stiffness of unextracted fibers were directly proportional to passive tension before activation. Similarly, passive stiffness due to weak bridges also increased linearly with passive tension, up to a limit. These correlations lead us to propose a stress-activation model for insect flight muscle in which passive tension is a prerequisite for the formation of both weak-binding and strong-binding cross-bridges.     

Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle

The effects of 2,3-butanedione 2-monoxime (BDM) on mechanical responses of glycerinated fibers and the ATPase activity of heavy meromyosin (HMM) and myofibrils have been studied using rabbit skeletal muscle. The mechanical responses and the ATPase activity were measured in similar conditions (ionic strength 0.06-0.2 M, 0.4-4 mM MgATP, 0-20 mM BDM, 2-20 degrees C and pH 7.0). BDM reversibly reduced the isometric tension, shortening speed, and instantaneous stiffness of the fibers. BDM also inhibited myofibrillar and HMM ATPase activities. The inhibitory effect on the relative ATPase activity of HMM was not influenced by the addition of actin or troponin-tropomyosin-actin. High temperature and low ionic strength weakened BDM's suppression of contraction of the fibers and the ATPase activity of contracting myofibrils, but not of the HMM, acto-HMM and relaxed myofibrillar ATPase activity. The size of the initial phosphate burst at 20 degrees C was independent of the concentration of BDM. These results suggest that the suppression of contraction of muscle fibers is due mainly to direct action of BDM on the myosin molecules.     

Tension transients initiated by photogeneration of MgADP in skinned skeletal muscle fibers

Addition of MgADP to skinned skeletal muscle fibers causes a rise in Ca(2+)-activated isometric tension. Mechanisms underlying this tension increase have been investigated by rapid photogeneration of ADP within skinned single fibers of rabbit psoas muscle. Photolysis of caged ADP (P2-1(2-nitrophenyl)ethyladenosine 5'-diphosphate) resulted in an exponential increase in isometric tension with an apparent rate constant, kADP, of 9.6 +/- 0.3 s-1 (mean +/- SE, n = 28) and an amplitude, PADP, of 4.9 +/- 0.3% Po under standard conditions (0.5 mM photoreleased MgADP, 4 mM MgATP, pH 7.0, pCa 4.5, 0.18 M ionic strength, 15 degrees C). PADP depended upon the concentration of photoreleased MgADP as well as the concentration of MgATP. A plot of 1/PADP vs. 1/[MgADP] at three MgATP concentrations was consistent with competition between MgADP and MgATP for the same site on the crossbridge. The rate of the transient, kADP, also depended upon the concentration of MgADP and MgATP. At both 4 and 1 mM MgATP, kADP was not significantly different after photorelease of 0.1-0.5 mM MgADP, but was reduced by 28-40% when 3.5 mM MgADP was added before photorelease of 0.5 mM MgADP. kADP was accelerated by about twofold when MgATP was varied from 0.5 to 8 mM MgATP. These effects of MgATP and MgADP were not readily accounted for by population of high force-producing states resulting from reversal of the ADP dissociation process. Rather, the results suggest that competition between MgADP and MgATP for crossbridges at the end of the cycle slows detachment leading to accumulation of force-generating crossbridges. Elevation of steady- state Pi concentration from 0.5 to 30 mM caused acceleration of kADP from 10.2 +/- 0.5 to 27.8 +/- 1.8 s-1, indicating that the tension rise involved crossbridge flux through the Pi dissociation step of the cycle.     

Sarcomeric disorganization in post-mortem fish muscles.

1. The post-mortem evolution of protein pattern in fish striated muscle was followed by SDS-PAGE, after different conditions of storage time and temperature. 2. Sarcoplasmic and sarcomeric fractions were analyzed respectively by low and high ionic strength extractions of fish muscle samples. 3. No evident modification of electrophoretic patterns was observed during the pre-rigor mortis period. 4. The high mol. wt proteins titin and nebulin were highly sensitive to proteolysis during the rigor mortis period. 5. Myosin extraction was predominantly influenced by the storage temperature. The myosin content of the extracts decreased during the rigor mortis period at storage temperatures greater than 8 degrees C. 6. alpha-Actinin was very resistant to proteolysis, but could be released from Z-disc structure during post-mortem aging.     

Nucleotide binding by myosin in glycerol-extracted rabbit skeletal muscle fibres at temperatures between -35 degrees C and 10 degrees C

Glycerol-extracted rabbit psoas fibres were incubated at temperatures between -35 degrees C and +10 degrees C in a low-ionic-strength relaxing solution containing 50% ethyleneglycol, 100 microM [3H]MgATP, 1 mM [14C]mannitol and less than 0.01 microM Ca2+. The fibres were then rinsed in a solution containing 1 mM ATP and the bound nucleotide eluted in trichloroacetic acid; all these operations were carried out at the cold temperature. Residual bound nucleotide was eluted with trichloroacetic acid at room temperature. The fibres were found to bind approximately 180 microM nucleotide, which is consistent with binding to the enzymatic site of myosin. The eluate, obtained in the cold, was analysed on poly(ethyleneimine)-cellulose for its ATP and ADP content. At temperatures down to -22 degrees C most of the bound nucleotide was ADP and there was little variation of this fraction with temperature. As the temperature was lowered below -22 degrees C the ATP fraction rose sharply; by -35 degrees C it predominated. These results are similar in type to those found by Biosca et al. [(1984) Biochemistry 23, 1947-1953] on isolated subfragment 1, but are displaced to a much lower temperature range. Thus in a muscle fibre only a low thermal energy is needed for myosin to hold its nucleotide in a constant balance between ATP and ADP.     

Passive tension and stiffness of vertebrate skeletal and insect flight muscles: the contribution of weak cross-bridges and elastic filaments.

Tension and dynamic stiffness of passive rabbit psoas, rabbit semitendinosus, and waterbug indirect flight muscles were investigated to study the contribution of weak-binding cross-bridges and elastic filaments (titin and minititin) to the passive mechanical behavior of these muscles. Experimentally, a functional dissection of the relative contribution of actomyosin cross-bridges and titin and minititin was achieved by 1) comparing mechanically skinned muscle fibers before and after selective removal of actin filaments with a noncalcium-requiring gelsolin fragment (FX-45), and 2) studying passive tension and stiffness as a function of sarcomere length, ionic strength, temperature, and the inhibitory effect of a carboxyl-terminal fragment of smooth muscle caldesmon. Our data show that weak bridges exist in both rabbit skeletal muscle and insect flight muscle at physiological ionic strength and room temperature. In rabbit psoas fibers, weak bridge stiffness appears to vary with both thin-thick filament overlap and with the magnitude of passive tension. Plots of passive tension versus passive stiffness are multiphasic and strikingly similar for these three muscles of distinct sarcomere proportions and elastic proteins. The tension-stiffness plot appears to be a powerful tool in discerning changes in the mechanical behavior of the elastic filaments. The stress-strain and stiffness-strain curves of all three muscles can be merged into one, by normalizing strain rate and strain amplitude of the extensible segment of titin and minititin, further supporting the segmental extension model of resting tension development.     

What limits the velocity of fast-skeletal muscle contraction in mammals?

In rat skeletal muscle the unloaded shortening velocity (Vo) is defined by the myosin isoform expressed in the muscle fibre. In 2001 we suggested that ADP release from actomyosin in solution (controlled by k(-AD)) was of the right size to limit Vo. However, to compare mechanical and solution kinetic data required a series of corrections to compensate for the differences in experimental conditions (0.5 M KCl, 22 degrees C for kinetic assays of myosin, 200 mM ionic strength, 12 degrees C to measure Vo). Here, a method was developed to prepare heavy meromyosin (HMM) from pure myosin isoforms isolated from single muscle fibres and to study k(-AD) (determined from the affinity of the acto-myosin complex for ADP, KAD) and the rate of ATP-induced acto-HMM dissociation (controlled by K1k+2) under the same experimental condition used to measure Vo). In fast-muscle myosin isolated from a wide range of mammalian muscles, k(-AD) was found to be too fast to limit Vo, whereas K1k+2 was of the right magnitude for ATP-induced dissociation of the cross-bridge to limit shortening velocity. The result was unexpected and prompted further experiments using the stopped-flow approach on myosin subfragment-1 (S1) and HMM obtained from bulk preparations of rabbit and rat muscle. These confirmed that the rate of cross-bridge dissociation by ATP limits the velocity of contraction for fast myosin II isoforms at 12 degrees C, while k(-AD) limits the velocity of slow myosin II isoforms. Extrapolating our data to 37 degrees C suggests that at physiological temperature the rate of ADP dissociation may limit Vo for both isoforms.     

Ca2+ dependence of the ATPase activity of phosphorylated smooth muscle myosin: effects of tropomyosin and actin

Calcium ions produce a 3-4-fold stimulation of the actin-activated ATPase activities of phosphorylated myosin from bovine pulmonary artery or chicken gizzard at 37 degrees C and at physiological ionic strengths, 0.12-0.16 M. Actins from either chicken gizzard or rabbit skeletal muscle stimulate the activity of phosphorylated myosin in a Ca2+-dependent manner, indicating that the Ca2+ sensitivity involves myosin or a protein associated with it. Partial loss of Ca2+ sensitivity upon treatment of phosphorylated gizzard myosin with low concentrations of chymotrypsin and the lack of any change on similar treatment of actin supports the above conclusion. Although both actins enhance ATPase activity, activation by gizzard actin exhibits Ca2+ dependence at higher temperatures or lower ionic strengths than does activation by skeletal muscle actin. The Ca2+ dependence of the activity of phosphorylated heavy meromyosin is about half that of myosin and is affected differently by temperature, ionic strength and Mg2+, being independent of temperature and optimal at lower concentrations of NaCl. Raising the concentration of Mg2+ above 2-3 mM inhibits the activity of heavy meromyosin but stimulates that of myosin, indicating that Mg2+ and Ca2+ activate myosin at different binding sites.     

X-ray diffraction studies of the thick filament in permeabilized myocardium from rabbit

Low angle x-ray diffraction patterns from relaxed permeabilized rabbit cardiac trabeculae and psoas muscle fibers were compared. Temperature was varied from 25 degrees C to 5 degrees C at 200 mM and 50 mM ionic strengths (mu), respectively. Effects of temperature and mu on the intensities of the myosin layer lines (MLL), the equatorial intensity ratio I(1,1)/I(1,0), and the spacing of the filament lattice are similar in both muscles. At 25 degrees C, particularly at mu = 50 mM, the x-ray patterns exhibited up to six orders of MLL and sharp meridional reflections, signifying that myosin heads (cross-bridges) are distributed in a well-ordered helical array. Decreasing temperature reduced MLL intensities but increased I(1,1)/I(1,0). Decreases in the MLL intensities indicate increasing disorder in the distribution of cross-bridges on the thick filaments surface. In the skeletal muscle, order/disorder is directly correlated with the hydrolysis equilibrium of ATP by myosin, [M.ADP.P(i)]/[M.ATP]. Similar effects of temperature on MLL and similar biochemical ATP hydrolysis pathway found in both types of muscles suggest that the order/disorder states of cardiac cross-bridges may well be correlated with the same biochemical and structural states. This implies that in relaxed cardiac muscle under physiological conditions, the unattached cross-bridges are largely in the M.ADP.P(i) state and with the lowering of the temperature, the equilibrium is increasingly in favor of [M.ATP] and [A.M.ATP]. There appear to be some differences in the diffraction patterns from the two muscles, however. Mainly, in the cardiac muscle, the MLL are weaker, the I(1,1)/I(1,0) ratio tends to be higher, and the lattice spacing D(10), larger. These differences are consistent with the idea that under a wide range of conditions, a greater fraction of cross-bridges is weakly bound to actin in the myocardium.     

Structures of actomyosin crossbridges in relaxed and rigor muscle fibers.

It was shown previously that a significant fraction of the myosin crossbridges is attached to actin in the skinned rabbit psoas fibers under relaxed conditions at low ionic strength and low temperature (Brenner, B., M. Schoenberg, J. M. Chalovich, L. E. Greene, and E. Eisenberg. 1982. Proc. Natl. Acad. Sci. USA. 79:7288-7291; Brenner, B., L. C. Lu, and R. J. Podolsky. 1984. Biophys. J. 46:299-306). In the present work, the structure of the attached crossbridges in the relaxed state between ionic strengths of 20 and 100 mM, as compared with that in the rigor state, is further examined by equatorial x-ray diffraction. Mass distributions projected along the fiber axis are reconstructed based on the first five equatorial reflections such that the spatial resolution is 128 A. The fraction of crossbridges attached under relaxed conditions are estimated to be in the range of 30% (at 100 mM ionic strength) and 60% (at 20 mM). The reconstructed density maps suggest that in the relaxed state, upon attachment the part of the crossbridge that centers around the thin filament is small, and the attachment does not significantly alter the center of mass of the myosin head distribution around the thick filament backbone. In contrast, accretion of mass in the rigor state occurs in a wider region surrounding the thin filament. In this case, mass in the surface region of the thick filament backbone is shifted slightly outward, probably by approximately 10 A. A schematic model for interpreting the present data is presented.     

Adsorption of glyceraldehyde 3-phosphate dehydrogenase on condensed monolayers of phospholipid.

The adsorption of [14C] alkylated glyceraldehyde 3-phosphate dehydrogenase from rabbit muscle to condensed monolayers of phosphatidic acid was investigated under a variety of conditions. 2. The rate constant for association at 20 degrees C depended on ionic strength. At I/2=60mM the rate constant was 0.39min-1. At I/2=260mM it decreased to 0.27min-1. 3. The apparent association constant (Kass.) for adsorption at I/2=60mM was 1.06 X 10(6)M-1 and was strongly influenced by subphase changes in pH and ionic strength. Measurements of Kass. at 20 degrees and 5 degrees C gave a value for the apparent enthalpy change on adsorption of -33kJ-mol-1. Calculations of the apparent change in free energy and apparent entropy change for the adsorption process gave values of -34kJ-mol-1 and +2J-K-1-mol-1 respectively. 4. Decreasing the amount of phosphatidic acid in the monolayer by replacement with phosphatidylcholine caused the shape of the adsorption isotherm to change from apparent hyperbolic to sigmoid. Subphase changes in pH or ionic strength did not affect the shape of the adsorption isotherm. However, adsorption of enzyme on monolayers of 100% phosphatidic acid in the presence of 1mM-CaCl2 was sigmoid in nature. 5. It is concluded that glyceraldehyde 3-phosphate dehydrogenase binds to condensed charged monolayers by multiple electrostatic interactions. At low concentrations of phosphatidic acid in the monolayer or in the presence of Ca2+, this occurs in a two-step process and depends on lateral diffusion of phosphatidic acid for strong binding to take place.     

Multiple folding pathways for the P4-P6 RNA domain

We recently described site-specific pyrene labeling of RNA to monitor Mg(2+)-dependent equilibrium formation of tertiary structure. Here we extend these studies to follow the folding kinetics of the 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA, using stopped-flow fluorescence with approximately 1 ms time resolution. Pyrene-labeled P4-P6 was prepared using a new phosphoramidite that allows high-yield automated synthesis of oligoribonucleotides with pyrene incorporated at a specific 2'-amino-2'-deoxyuridine residue. P4-P6 forms its higher-order tertiary structure rapidly, with k(obs) = 15-31 s(-1) (t(1/2) approximately 20-50 ms) at 35 degrees C and [Mg(2+)] approximately 10 mM in Tris-borate (TB) buffer. The folding rate increases strongly with temperature from 4 to 45 degrees C, demonstrating a large activation enthalpy DeltaH(double dagger) approximately 26 kcal/mol; the activation entropy DeltaS(double dagger) is large and positive. In low ionic strength 10 mM sodium cacodylate buffer at 35 degrees C, a slow (t(1/2) approximately 1 s) folding component is also observed. The folding kinetics are both ionic strength- and temperature-dependent; the slow phase vanishes upon increasing [Na(+)] in the cacodylate buffer, and the kinetics switch completely from fast at 30 degrees C to slow at 40 degrees C. Using synchrotron hydroxyl radical footprinting, we confirm that fluorescence monitors the same kinetic events as hydroxyl radical cleavage, and we show that the previously reported slow P4-P6 folding kinetics apply only to low ionic strength conditions. One model to explain the fast and slow folding kinetics postulates that some tertiary interactions are present even without Mg(2+) in the initial state. The fast kinetic phase reflects folding that is facilitated by these interactions, whereas the slow kinetics are observed when these interactions are disrupted at lower ionic strength and higher temperature.     

X-ray diffraction and electron microscopy from Lethocerus flight muscle partially relaxed by adenylylimidodiphosphate and ethylene glycol

The low-angle X-ray diffraction pattern from Lethocerus flight muscle fibres was recorded in rigor or under two conditions that modify crossbridge structure and behaviour, aqueous adenylylimidodiphosphate (AMPPNP) and AMPPNP + calcium in an ethylene glycol-water mixture. The effects on the 38.7 nm layer-line peaks (hk.6) of the diffraction patterns were studied in detail. In aqueous AMPPNP at room temperature, a condition in which rigor tension drops to half without loss of stiffness, the peaks remained nearly as intense as in rigor except for the 10.6, which dropped to half. In 20% (v/v) ethylene glycol-AMPPNP + 100 microM-Ca2+ at 23 degrees C (gly + pnp + Ca), a condition which removed muscle tension but left stiffness close to the rigor value, the 10.6 and 11.6 peaks greatly decreased but the 31.6 remained relatively high. The 14.5 nm meridional peak (00.16) became stronger on addition of AMPPNP and again on adding glycol + calcium. Considered in terms of constructively interfering filaments and crossbridges, the X-ray data indicated a transfer of diffracting crossbridge mass towards the thick filament as relaxation proceeds. We compared the X-ray diffraction patterns and crossbridge structure seen with electron microscopy (EM) under the same chemical conditions. EM and X-ray observations were mutually quite consistent overall. However, X-ray data indicated that more crossbridge mass was stereospecifically related to actin before fixation in the partially relaxed state (gly + pnp + Ca) than was suggested by the disordered crossbridge profiles seen by EM. We conclude that myosin heads at the start of the power stroke may both be closely related to their thick filament origins and form actin-determined attachments to the thin filament.