Effect of inorganic phosphate on the force and number of myosin cross-bridges during the isometric contraction of permeabilized muscle fibers from rabbit psoas

The relation between the chemical and mechanical steps of the myosin-actin ATPase reaction that leads to generation of isometric force in fast skeletal muscle was investigated in demembranated fibers of rabbit psoas muscle by determining the effect of the concentration of inorganic phosphate (Pi) on the stiffness of the half-sarcomere (hs) during transient and steady-state conditions of the isometric contraction (temperature 12°C, sarcomere length 2.5 μm). Changes in the hs strain were measured by imposing length steps or small 4 kHz oscillations on the fibers in control solution (without added Pi) and in solution with 3-20 mM added Pi. At the plateau of the isometric contraction in control solution, the hs stiffness is 22.8 ± 1.1 kPa nm⁻¹. Taking the filament compliance into account, the total stiffness of the array of myosin cross-bridges in the hs (e) is 40.7 ± 3.7 kPa nm⁻¹. An increase in [Pi] decreases the stiffness of the cross-bridge array in proportion to the isometric force, indicating that the force of the cross-bridge remains constant independently of [Pi]. The rate constant of isometric force development after a period of unloaded shortening (r_F) is 23.5 ± 1.0 s⁻¹ in control solution and increases monotonically with [Pi], attaining a maximum value of 48.6 ± 0.9 s⁻¹ at 20 mM [Pi], in agreement with the idea that Pi release is a relatively fast step after force generation by the myosin cross-bridge. During isometric force development at any [Pi], e and thus the number of attached cross-bridges increase in proportion to the force, indicating that, independently of the speed of the process that leads to myosin attachment to actin, there is no significant (>1 ms) delay between generation of stiffness and generation of force by the cross-bridges.     

The Effect of Myofilament Compliance on Kinetics of Force Generation by Myosin Motors in Muscle

We use the inhibitor of isometric force of skeletal muscle N-benzyl-p-toluene sulfonamide (BTS) to decrease, in a dose dependent way, the number of myosin motors attached to actin during the steady isometric contraction of single fibers from frog skeletal muscle (4°C, 2.1 μm sarcomere length). In this way we can reduce the strain in the myofilament compliance during the isometric tetanus (T₀) from 3.54 nm in the control solution (T_0,NR) to ∼0.5 nm in 1 μM BTS, where T₀ is reduced to ∼0.15 T_0,NR. The quick force recovery after a step release (1-3 nm per half-sarcomere) becomes faster with the increase of BTS concentration and the decrease of T₀. The simulation of quick force recovery with a multistate model of force generation, that adapts Huxley and Simmons model to account for both the high stiffness of the myosin motor (∼3 pN/nm) and the myofilament compliance, shows that the increase in the rate of quick force recovery by BTS is explained by the reduced strain in the myofilaments, consequent to the decrease in half-sarcomere force. The model estimates that i), for the same half-sarcomere release the state transition kinetics in the myosin motor are five times faster in the absence of filament compliance than in the control; and ii), the rate of force recovery from zero to T₀ is ∼6000/s in the absence of filament compliance.     

On the mechanical stabilization of filopodia

We studied force-induced elongation of filopodia by coupling magnetic tweezers to the tip through the bacterial coat protein invasin, which couples the force generator to the actin bundles (through myosin X), thus impeding the growth of the actin plus end. Single force pulses (15–30 s) with amplitudes between 20 and 600 pN and staircase-like force scenarios (amplitudes, ∼50 pN; step widths, 30 s) were applied. In both cases, the responses consist of a fast viscoelastic deflection followed by a linear flow regime. The deflections are reversible after switching off the forces, suggesting a mechanical memory. The elongation velocity exhibits an exponential distribution (half-width <v_1/2>, ∼0.02 μm s⁻¹) and did not increase systematically with the force amplitudes. We estimate the bending modulus (0.4 × 10⁻²³ J m) and the number of actin filaments (∼10) by analyzing filopodium bending fluctuations. Sequestering of intracellular Ca²⁺ by BAPTA caused a strong reduction in the amplitude of elongation, whereas latrunculin A resulted in loss of the elastic response. We attribute the force-independent velocity to the elongation of actin bundles enabled by the force-induced actin membrane uncoupling and the reversibility by the treadmilling mechanism and an elastic response.     

Reverse Conformational Changes of the Light Chain-Binding Domain of Myosin V and VI Processive Motor Heads during and after Hydrolysis of ATP by Small-Angle X-Ray Solution Scattering

We used small-angle X-ray solution scattering (SAXS) technique to investigate the nucleotide-mediated conformational changes of the head domains [subfragment 1 (S1)] of myosin V and VI processive motors that govern their directional preference for motility on actin. Recombinant myosin V-S1 with two IQ motifs (MV-S1IQ2) and myosin VI-S1 (MVI-S1) were engineered from Sf9 cells using a baculovirus expression system. The radii of gyration (R_g) of nucleotide-free MV-S1IQ2 and MVI-S1 were 48.6 and 48.8 Å, respectively. In the presence of ATP, the R_g value of MV-S1IQ2 decreased to 46.7 Å, while that of MVI-S1 increased to 51.7 Å, and the maximum chord length of the molecule decreased by ca 9% for MV-S1IQ2 and increased by ca 6% for MVI-S1. These opposite directional changes were consistent with those occurring in S1s with ADP and Vi or AlF₄^− 2 bound (i.e., in states mimicking the ADP/Pi-bound state of ATP hydrolysis). Binding of AMPPNP induced R_g changes of both constructs similar to those in the presence of ATP, suggesting that the timing of the structural changes for their motion on actin is upon binding of ATP (the pre-hydrolysis state) during the ATPase cycle. Binding of ADP to MV-S1IQ2 and MVI-S1 caused their R_g values to drop below those in the nucleotide-free state. Thus, upon the release of Pi, the reverse conformational change could occur, coupling to drive the directional motion on actin. The amount of R_g change upon the release of Pi was ca 6.4 times greater in MVI-S1 than in MV-S1IQ2, relating to the production of the large stroke of the MVI motor during its translocation on actin. Atomic structural models for these S1s based upon the ab initio shape reconstruction from X-ray scattering data were constructed, showing that MVI-S1 has the light-chain-binding domain positioned in the opposite direction to MV-S1IQ2 in both the pre- and post-powerstroke transition. The angular change between the light chain-binding domains of MV-S1IQ2 in the pre- to post-powerstroke transition was ∼ 50°, comparable to that of MII-S1. On the other hand, that of MVI-S1 was ∼ 100° or ∼ 130° much less than the currently postulated changes to allow the maximal stroke size of myosin VI-S1 but still significantly larger than those of other myosins reported so far. The results suggest that some additional alterations or elements are required for MVI-S1 to take maximal working strokes along the actin filament.     

Time Course and Strain Dependence of ADP Release during Contraction of Permeabilized Skeletal Muscle Fibers

A phosphorylated, single cysteine mutant of nucleoside diphosphate kinase, labeled with N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (P∼NDPK-IDCC), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during contraction of single permeabilized rabbit psoas fibers. The dephosphorylation of the phosphorylated protein by MgADP occurs within the lattice environment of permeabilized fibers with a second-order rate constant at 12°C of 10⁵ M⁻¹ s⁻¹. This dephosphorylation is accompanied by a change in coumarin fluorescence. We report the time course of P∼NDPK-IDCC dephosphorylation during the period of active isometric force redevelopment after quick release of fiber strain at pCa²⁺ of 4.5. After a rapid length decrease of 0.5% was applied to the fiber, the extra NDPK-IDCC produced during force recovery, above the value during the approximately steady state of isometric contraction, was 2.7 ± 0.6 μM and 4.7 ± 1.5 μM at 12 and 20°C, respectively. The rates of P∼NDPK-IDCC dephosphorylation during force recovery were 28 and 50 s⁻¹ at 12 and 20°C, respectively. The time courses of isometric force and P∼NDPK-IDCC dephosphorylation were simulated using a seven-state reaction scheme. Relative isometric force was modeled by changes in the occupancy of strongly bound A.M.ADP.P_i and A.M.ADP states. A strain-sensitive A.M.ADP isomerization step was rate-limiting (3-6 s⁻¹) in the cross-bridge turnover during isometric contraction. At 12°C, the A.M.ADP.P_i and the pre- and postisomerization A.M.ADP states comprised 56%, 38%, and 7% of the isometric force-bearing AM states, respectively. At 20°C, the force-bearing A.M.ADP.P_i state was a lower proportion of the total force-bearing states (37%), whereas the proportion of postisomerization A.M.ADP states was higher (19%). The simulations suggested that release of cross-bridge strain caused rapid depopulation of the preisomerization A.M.ADP state and transient accumulation of MgADP in the postisomerization A.M.ADP state. Hence, the strain-sensitive isomerization of A.M.ADP seems to explain the rate of change of P∼NDPK-IDCC dephosphorylation during force recovery. The temperature-dependent isometric distribution of myosin states is consistent with the previous observation of a small decrease in amplitude of the P_i transient during force recovery at 20°C and the current observation of an increase in amplitude of the ADP-sensitive NDPK-IDCC transient.     

Direct Observation of Phosphate Inhibiting the Force-Generating Capacity of a Miniensemble of Myosin Molecules

Elevated levels of phosphate (P_i) reduce isometric force, providing support for the notion that the release of P_i from myosin is closely associated with the generation of muscular force. P_i is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of P_i on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of P_i on the force-generating capacity of a small ensemble of myosin (∼12 myosin heads) using a three-bead laser trap assay. In the absence of P_i, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM P_i, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated P_i also caused a >65% reduction in binding-event lifetime, suggesting that P_i induces premature detachment from a strongly bound state. Definitive evidence of a P_i-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which P_i induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of P_i on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that P_i-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.     

The effect of ATP analogs on posthydrolytic and force development steps in skinned skeletal muscle fibers.

ATP, 2-deoxy ATP (dATP), CTP, and UTP support isometric force and unloaded shortening velocity (Vu) to various extents (Regnier et al., Biophys. J. 74:3044-3058). Vu correlated with the rate of cross-bridge dissociation after the power stroke and the steady-state hydrolysis rate in solution, whereas force was modulated by NTP binding and cleavage. Here we studied the influence of posthydrolytic cross-bridge steps on force and fiber shortening by measuring isometric force and stiffness, the rate of tension decline (kPi) after Pi photogeneration from caged Pi, and the rate of tension redevelopment (ktr) after a sudden release and restretch of fibers. The slope of the force versus [Pi] relationship was the same for ATP, dATP, and CTP, but for UTP it was threefold less. ktr and kPi increased with increasing [Pi] with a similar slope for ATP, dATP, and CTP, but had an increasing magnitude of the relationship ATP < dATP < CTP. UTP reduced ktr but increased kPi. The results suggest that the rate constant for the force-generating isomerization increases with the order ATP < dATP < CTP < UTP. Simulations using a six-state model suggest that increasing the force-generating rate accounts for the faster kPi in dATP, CTP, and UTP. In contrast, ktr appears to be strongly affected by the rates of NTP binding and cleavage and the rate of the force-generating isomerization.     

Induction of G-quadruplex DNA structure by Zn(II) 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin

G-quadruplexes (GQ) are formed by the association of guanine-rich stretches of DNA. Certain small molecules can influence kinetics and thermodynamics of this association. Understanding the mechanism of ligand-assisted GQ folding is necessary for the design of more efficient cancer therapeutics. The oligonucleotide d(TAGGG)₂ forms parallel bimolecular GQ in the presence of ≥66 mM K⁺; GQs are not formed under Na⁺, Li⁺ or low K⁺ conditions. The thermodynamic parameters for GQ folding at 60 μM oligonucleotide and 100 mM KCl are ΔH = −35 ± 2 kcal mol⁻¹ and ΔG₃₁₀ = −1.4 kcal mol⁻¹. Quadruplex [d(TAGGG)₂]₂ binds 2-3 K⁺ ions with K_d of 0.5 ± 0.2 mM. Our work addresses the question of whether metal free 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) and its Zn(II), Cu(II), and Pt(II) derivatives are capable of facilitating GQ folding of d(TAGGG)₂ from single stranded, or binding to preformed GQ, using UV-vis and circular dichroism (CD) spectroscopies. ZnTMPyP4 is unique among other porphyrins in its ability to induce GQ structure of d(TAGGG)₂, which also requires at least a low amount of potassium. ZnTMPyP4 binds with 2:1 stoichiometry possibly in an end-stacking mode with a ∼10⁶ M⁻¹ binding constant, determined through UV-vis and ITC titrations. This process is entropically driven and has ΔG₂₉₈ of −8.0 kcal mol⁻¹. TMPyP4 binds with 3:1 stoichiometry and K_a of ∼10⁶ M⁻¹. ZnTMPyP4 and TMPyP4 are efficient stabilizers of [d(TAGGG)₂]₂ displaying ΔT_1/2 of 13.5 and 13.8 °C, respectively, at 1:2 GQ to porphyrin ratio; CuTMPyP4 shows a much weaker effect (ΔT_1/2 = 4.7 °C) and PtTMPyP4 is weakly destabilizing (ΔT_1/2 = −2.9 °C). The selectivity of ZnTMPyP4 for GQ versus dsDNA is comparable to that of TMPyP4. The ability of ZnTMPyP4 to bind and stabilize GQ, to induce GQ formation, and speed up its folding may suggest an important biological activity for this molecule.     

Enhanced active cross-bridges during diastole: molecular pathogenesis of tropomyosin's HCM mutations

Three HCM-causing tropomyosin (Tm) mutants (V95A, D175N, and E180G) were examined using the thin-filament extraction and reconstitution technique. The effects of Ca²⁺, ATP, phosphate, and ADP concentrations on cross-bridge kinetics in myocardium reconstituted with each of these mutants were studied at 25°C, and compared to wild-type (WT) Tm at physiological ionic strength (200 mM). All three mutants showed significantly higher (2–3.5 fold) low Ca²⁺ tension (T_LC) and stiffness than WT at pCa 8.0. High Ca²⁺ tension (T_HC) was significantly higher for E180G than that for WT, whereas T_HC of V95A and D175N was similar to WT; high Ca²⁺ stiffness (Y_HC) had the same trend. The Ca²⁺ sensitivity of isometric force was significantly greater for V95A and E180G than for WT, whereas that of D175N remained the same as for WT; for all mutants, cooperativity was lower than for WT. Nine kinetic constants and the cross-bridge distribution were deduced using sinusoidal analysis. The number of force-generating cross bridges was similar among the D175N, E180G, and WT Tm forms, but it was significantly larger in the case of V95A than WT. We conclude that the increased number of actively cycling cross bridges at pCa 8 is the major cause of Tm mutation-related HCM pathogenesis, which may result in diastolic dysfunction. Decreased contractility (T_act) in V95A and D175N may further contribute to the severity of myocyte hypertrophy and related prognosis of the disease.     

Coupling between phosphate release and force generation in muscle actomyosin

Energetic, kinetic and oxygen exchange experiments in the mid-1980s and early 1990s suggested that phosphate (Pi) release from actomyosin-adenosine diphosphate Pi (AM.ADP.Pi) in muscle fibres is linked to force generation and that Pi release is reversible. The transition leading to the force-generating state and subsequent Pi release were hypothesized to be separate, but closely linked steps. Pi shortens single force-generating actomyosin interactions in an isometric optical clamp only if the conditions enable them to last 20-40 ms, enough time for Pi to dissociate. Until 2003, the available crystal forms of myosin suggested a rigid coupling between movement of switch II and tilting of the lever arm to generate force, but they did not explain the reciprocal affinity myosin has for actin and nucleotides. Newer crystal forms and other structural data suggest that closing of the actin-binding cleft opens switch I (presumably decreasing nucleotide affinity). These data are all consistent with the order of events suggested before: myosin.ADP.Pi binds weakly, then strongly to actin, generating force. Then Pi dissociates, possibly further increasing force or sliding.     

A kinetic model that explains the effect of inorganic phosphate on the mechanics and energetics of isometric contraction of fast skeletal muscle

A conventional five-step chemo-mechanical cycle of the myosin–actin ATPase reaction, which implies myosin detachment from actin upon release of hydrolysis products (ADP and phosphate, Pi) and binding of a new ATP molecule, is able to fit the [Pi] dependence of the force and number of myosin motors during isometric contraction of skeletal muscle. However, this scheme is not able to explain why the isometric ATPase rate of fast skeletal muscle is decreased by an increase in [Pi] much less than the number of motors. The question can be solved assuming the presence of a branch in the cycle: in isometric contraction, when the force generation process by the myosin motor is biased at the start of the working stroke, the motor can detach at an early stage of the ATPase cycle, with Pi still bound to its catalytic site, and then rapidly release the hydrolysis products and bind another ATP. In this way, the model predicts that in fast skeletal muscle the energetic cost of isometric contraction increases with [Pi]. The large dissociation constant of the product release in the branched pathway allows the isometric myosin–actin reaction to fit the equilibrium constant of the ATPase.     

Direct Observation of Phosphate Inhibiting the Force-Generating Capacity of a Miniensemble of Myosin Molecules

Elevated levels of phosphate (P_i) reduce isometric force, providing support for the notion that the release of P_i from myosin is closely associated with the generation of muscular force. P_i is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of P_i on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of P_i on the force-generating capacity of a small ensemble of myosin (∼12 myosin heads) using a three-bead laser trap assay. In the absence of P_i, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM P_i, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated P_i also caused a >65% reduction in binding-event lifetime, suggesting that P_i induces premature detachment from a strongly bound state. Definitive evidence of a P_i-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which P_i induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of P_i on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that P_i-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.     

Measuring kinetic dissociation/association constants between Lactococcus lactis bacteria and mucins using living cell probes

In this work we focused on quantifying adhesion between Lactococcus lactis, the model for lactic acid bacteria (LAB) and mucins. Interactions between two strains of L. lactis (IBB477 and MG1820 as control) and pig gastric mucin–based coating were measured and compared with the use of atomic force microscopy. Analysis of retraction force-distance curves shed light on the differential contributions of nonspecific and specific forces. An increased proportion of specific adhesive events was obtained for IBB477 (20% vs. 5% for the control). Blocking assays with free pig gastric mucin and its O-glycan moiety showed that oligosaccharides play a major (but not exclusive) role in L. lactis-mucins interactions. Specific interactions were analyzed in terms of kinetic constants. An increase in the loading rate of atomic force microscope tip led to a higher force between interacting biological entities, which was directly linked to the kinetic dissociation constant (K_off). Enhancing the contact time between the tip and the sample allowed an increase in the interaction probability, which can be related to the kinetic association constant (K_on). Variations in the loading rate and contact time enabled us to determine K_on (3.3 × 10² M⁻¹·s⁻¹) and K_off (0.46 s⁻¹), and the latter was consistent with values given in the literature for sugar-protein interactions.     

Cooperative Cross-Bridge Activation of Thin Filaments Contributes to the Frank-Starling Mechanism in Cardiac Muscle

Myosin cross-bridges play an important role in the regulation of thin-filament activation in cardiac muscle. To test the hypothesis that sarcomere length (SL) modulation of thin-filament activation by strong-binding cross-bridges underlies the Frank-Starling mechanism, we inhibited force and strong cross-bridge binding to intermediate levels with sodium vanadate (Vi). Force and stiffness varied proportionately with [Ca²⁺] and [Vi]. Increasing [Vi] (decreased force) reduced the pCa₅₀ of force-[Ca²⁺] relations at 2.3 and 2.0 μm SL, with little effect on slope (n_H). When maximum force was inhibited to ∼40%, the effects of SL on force were diminished at lower [Ca²⁺], whereas at higher [Ca²⁺] (pCa < 5.6) the relative influence of SL on force increased. In contrast, force inhibition to ∼20% significantly reduced the sensitivity of force-[Ca²⁺] relations to changes in both SL and myofilament lattice spacing. Strong cross-bridge binding cooperatively induced changes in cardiac troponin C structure, as measured by dichroism of 5′ iodoacetamido-tetramethylrhodamine-labeled cardiac troponin C. This apparent cooperativity was reduced at shorter SL. These data emphasize that SL and/or myofilament lattice spacing modulation of the cross-bridge component of cardiac thin-filament activation contributes to the Frank-Starling mechanism.     

Cardiac Myosin-binding protein C modulates the tuning of the molecular motor in the heart

Cardiac myosin binding protein C (cMyBP-C) is an important regulator of cardiac contractility. Its precise effect on myosin cross-bridges (CBs) remains unclear. Using a cMyBP-C^−/− mouse model, we determined how cMyBP-C modulates the cyclic interaction of CBs with actin. From papillary muscle mechanics, CB characteristics were provided using A. F. Huxley's equations. The probability of myosin being weakly bound to actin was higher in cMyBP-C^−/− than in cMyBP-C^+/+. However, the number of CBs in strongly bound, high-force generated state and the force generated per CB were lower in cMyBP-C^−/−. Overall CB cycling and the velocity of CB tilting were accelerated in cMyBP-C^−/−. Taking advantage of the presence of cMyBP-C in cMyBP-C^+/+ myosin solution but not in cMyBP-C^−/−, we also analyzed the effects of cMyBP-C on the myosin-based sliding velocity of actin filaments. At baseline, sliding velocity and the relative isometric CB force, as determined by the amount of α-actinin required to arrest thin filament motility, were lower in cMyBP-C^−/− than in cMyBP-C^+/+. cAMP-dependent protein kinase-mediated cMyBP-C phosphorylation further increased the force produced by CBs. We conclude that cMyBP-C prevents inefficient, weak binding of the myosin CB to actin and has a critical effect on the power-stroke step of the myosin molecular motor.     

Kinetic mechanism of the Ca2+-dependent switch-on and switch-off of cardiac troponin in myofibrils

The kinetics of Ca²⁺-dependent conformational changes of human cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cysteine 84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole and reconstituted with cTnI and cTnT to the cTn complex, which was incorporated into guinea pig cardiac myofibrils. These exchanged myofibrils, or the isolated cTn, were rapidly mixed in a stopped-flow apparatus with different [Ca²⁺] or the Ca²⁺-buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid to determine the kinetics of the switch-on or switch-off, respectively, of cTn. Activation of myofibrils with high [Ca²⁺] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s⁻¹ and ∼330 s⁻¹, respectively. At low [Ca²⁺] (pCa 6.6), the slower rate was reduced to ∼25 s⁻¹, but was still ∼50-fold higher than the rate constant of Ca²⁺-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca²⁺] from pCa 5.0-7.9 induced a fluorescence decay with a rate constant of 39 s⁻¹, which was approximately fivefold faster than force relaxation. Modeling the data indicates two sequentially coupled conformational changes of cTnC in myofibrils: 1), rapid Ca²⁺-binding (k_B ≈ 120 μM⁻¹ s⁻¹) and dissociation (k_D ≈ 550 s⁻¹); and 2), slower switch-on (k_on = 390s⁻¹) and switch-off (k_off = 36s⁻¹) kinetics. At high [Ca²⁺], ∼90% of cTnC is switched on. Both switch-on and switch-off kinetics of incorporated cTn were around fourfold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and directly rate-limit neither cardiac myofibrillar contraction nor relaxation.     

Synchronous In Situ ATPase Activity, Mechanics, and Ca Sensitivity of Human and Porcine Myocardium

Flash-frozen myocardium samples provide a valuable means of correlating clinical cardiomyopathies with abnormalities in sarcomeric contractile and biochemical parameters. We examined flash-frozen left-ventricle human cardiomyocyte bundles from healthy donors to determine control parameters for isometric tension (P_o) development and Ca²⁺ sensitivity, while simultaneously measuring actomyosin ATPase activity in situ by a fluorimetric technique. P_o was 17 kN m⁻² and pCa_50% was 5.99 (28°C, I = 130 mM). ATPase activity increased linearly with tension to 132 μM s⁻¹. To determine the influence of flash-freezing, we compared the same parameters in both glycerinated and flash-frozen porcine left-ventricle trabeculae. P_o in glycerinated porcine myocardium was 25 kN m⁻², and maximum ATPase activity was 183 μM s⁻¹. In flash-frozen porcine myocardium, P_o was 16 kN m⁻² and maximum ATPase activity was 207 μM s⁻¹. pCa_50% was 5.77 in the glycerinated and 5.83 in the flash-frozen sample. Both passive and active stiffness of flash-frozen porcine myocardium were lower than for glycerinated tissue and similar to the human samples. Although lower stiffness and isometric tension development may indicate flash-freezing impairment of axial force transmission, we cannot exclude variability between samples as the cause. ATPase activity and pCa_50% were unaffected by flash-freezing. The lower ATPase activity measured in human tissue suggests a slower actomyosin turnover by the contractile proteins.     

Unfolding thermodynamics of the Delta-domain in the prohead I subunit of phage HK97: determination by factor analysis of Raman spectra

An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (∼ 70%), median melting temperature (T_m = 58 °C), enthalpy (ΔH_m = 50 ± 5 kcal mol^− 1), entropy (ΔS_m = 150 ± 10 cal mol^− 1 K^− 1), and average cooperative melting unit (〈n_c〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability (T_m = 40 °C; ΔH_m = 27 ± 2 kcal mol^− 1; ΔS_m = 86 ± 6 cal mol^− 1 K^− 1) and noncooperative unfolding (〈n_c〉 ∼ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.     

Interaction of minor groove ligands with G-quadruplexes: thermodynamic contributions of the number of quartets, T-U substitutions, and conformation

In the presence of specific metal ions, DNA oligonucleotides containing guanine repeat sequences can adopt G-quadruplex structures. In this work, we used a combination of spectroscopic and calorimetric techniques to investigate the conformation and unfolding thermodynamics of the K⁺-form of five G-quadruplexes with sequences: d(G₂T₂G₂TGTG₂T₂G₂), G2, d(G₃T₂G₃TGTG₃T₂G₃), G3, their analogs where T is replaced with U, G2-U and G3-U, and r(G₂U₂G₂UGUG₂U₂G₂), rG2. These G-quadruplexes show CD spectra characteristic of the “chair” conformation (G2 and G2-U), or “basket” conformation (rG2); or a mixture of these two conformers (G3 and G3-U). Thermodynamic profiles show that the favorable folding of each G-quadruplex results from the typical compensation of a favorable enthalpy and unfavorable entropy contributions. G-quadruplex stability increase in the following order (in ΔG°₂₀): rG2 (−1.3 kcal/mol) < G2 < G2-U <G3-U (chair) < G3 (chair) <G3-U (basket) < G3 (basket) (−8.6 kcal/mol), due to favorable enthalpy contribution from the stacking of G-quartets.We used ITC to determine thermodynamic binding profiles for the interaction of the minor groove ligands, netropsin and distamycin, with each G-quadruplex. Both ligands bind with high exothermic enthalpies (∼−10.8 kcal/mol), 1:1 stoichiometries, and weak affinities (∼8 × 10⁴ M⁻¹). The similarity of the binding thermodynamic profiles, together with the absence of induced Cotton effects, indicates a surface or outside binding mode. We speculate that the top and bottom surfaces of the G-quadruplex comprise the potential MGBL binding sites, where the ligand lies on the surface forming van der Waals interactions with the guanines of the G-quartets and loop nucleotides.     

The Fraction of Myosin Motors That Participate in Isometric Contraction of Rabbit Muscle Fibers at Near-Physiological Temperature

The duty ratio, or the part of the working cycle in which a myosin molecule is strongly attached to actin, determines motor processivity and is required to evaluate the force generated by each molecule. In muscle, it is equal to the fraction of myosin heads that are strongly, or stereospecifically, bound to the thin filaments. Estimates of this fraction during isometric contraction based on stiffness measurements or the intensities of the equatorial or meridional x-ray reflections vary significantly. Here, we determined this value using the intensity of the first actin layer line, A1, in the low-angle x-ray diffraction patterns of permeable fibers from rabbit skeletal muscle. We calibrated the A1 intensity by considering that the intensity in the relaxed and rigor states corresponds to 0% and 100% of myosin heads bound to actin, respectively. The fibers maximally activated with Ca²⁺ at 4°C were heated to 31–34°C with a Joule temperature jump (T-jump). Rigor and relaxed-state measurements were obtained on the same fibers. The intensity of the inner part of A1 during isometric contraction compared with that in rigor corresponds to 41–43% stereospecifically bound myosin heads at near-physiological temperature, or an average force produced by a head of ∼6.3 pN.