Effects of Hydrostatic-Pressure on Muscle Contraction: A Look Back on Some Experimental Findings.

Findings from experiments that used hydrostatic pressure changes to analyse the process of skeletal muscle contraction are re-examined. The force in resting muscle is insensitive to an increase in hydrostatic pressure from 0.1 MPa (atmospheric) to 10 MPa, as also found for force in rubber-like elastic filaments. The force in rigour muscle rises with increased pressure, as shown experimentally for normal elastic fibres (e.g., glass, collagen, keratin, etc.). In submaximal active contractions, high pressure leads to tension potentiation. The force in maximally activated muscle decreases with increased pressure: the extent of this force decrease in maximal active muscle is sensitive to the concentration of products of ATP hydrolysis (Pi-inorganic phosphate and ADP-adenosine diphosphate) in the medium. When the increased hydrostatic pressure is rapidly decreased, the force recovered to the atmospheric level in all cases. Thus, the resting muscle force remained the same: the force in the rigour muscle decreased in one phase and that in active muscle increased in two phases. The rate of rise of active force on rapid pressure release increased with the concentration of Pi in the medium, indicating that it is coupled to the Pi release step in the ATPase-driven crossbridge cycle in muscle. Pressure experiments on intact muscle illustrate possible underlying mechanisms of tension potentiation and causes of muscle fatigue.

MyBP-C: one protein to govern them all.

The heart is an extraordinarily versatile pump, finely tuned to respond to a multitude of demands. Given the heart pumps without rest for decades its efficiency is particularly relevant. Although many proteins in the heart are essential for viability, the non-essential components can attract numerous mutations which can cause disease, possibly through alterations in pumping efficiency. Of these, myosin binding protein C is strongly over-represented with ~ 40% of all known mutations in hypertrophic cardiomyopathy. Therefore, a complete understanding of its molecular function in the cardiac sarcomere is warranted. In this review, we revisit contemporary and classical literature to clarify both the current standing of this fast-moving field and frame future unresolved questions. To date, much effort has been directed at understanding MyBP-C function on either thick or thin filaments. Here we aim to focus questions on how MyBP-C functions at a molecular level in the context of both the thick and thin filaments together. A concept that emerges is MyBP-C acts to govern interactions on two levels; controlling myosin access to the thin filament by sequestration on the thick filament, and controlling the activation state and access of myosin to its binding sites on the thin filament. Such affects are achieved through directed interactions mediated by phosphorylation (of MyBP-C and other sarcomeric components) and calcium.

Towards a unified theory of muscle contraction. I: foundations.

Molecular models of contractility in striated muscle require an integrated description of the action of myosin motors, firstly in the filament lattice of the half-sarcomere. Existing models do not adequately reflect the biochemistry of the myosin motor and its sarcomeric environment. The biochemical actin-myosin-ATP cycle is reviewed, and we propose a model cycle with two 4- to 5-nm working strokes, where phosphate is released slowly after the first stroke. A smaller third stroke is associated with ATP-induced detachment from actin. A comprehensive model is defined by applying such a cycle to all myosin-S1 heads in the half-sarcomere, subject to generic constraints as follows: (a) all strain-dependent kinetics required for actin-myosin interactions are derived from reaction-energy landscapes and applied to dimeric myosin, (b) actin-myosin interactions in the half-sarcomere are controlled by matching rules derived from the structure of the filaments, so that each dimer may be associated with a target zone of three actin sites, and (c) the myosin and actin filaments are treated as elastically extensible. Numerical predictions for such a model are presented in the following paper.

Kinetic analysis of the slow skeletal myosin MHC-1 isoform from bovine masseter muscle.

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as beta-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin-ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant K(D)) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (K(AD)/K(D) approximately 5 instead of K(AD)/K(D) approximately 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s(-1)) than reported previously (138 s(-1)). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.

Molecular mechanism of actomyosin-based motility.

Sophisticated molecular genetic, biochemical and biophysical studies have been used to probe the molecular mechanism of actomyosin-based motility. Recent solution measurements, high-resolution structures of recombinant myosin motor domains, and lower resolution structures of the complex formed by filamentous actin and the myosin motor domain provide detailed insights into the mechanism of chemomechanical coupling in the actomyosin system. They show how small conformational changes are amplified by a lever-arm mechanism to a working stroke of several nanometres, explain the mechanism that governs the directionality of actin-based movement, and reveal a communication pathway between the nucleotide binding pocket and the actin-binding region that explains the reciprocal relationship between actin and nucleotide affinity. Here we focus on the interacting elements in the actomyosin system and the communication pathways in the myosin motor domain that respond to actin binding.

Ionic interactions play a role in the regulatory mechanism of scallop heavy meromyosin.

Heavy meromyosin from scallop (scHMM) striated muscle is regulated by calcium binding to the essential light chain. The regulation can be modeled with a calcium-dependent equilibrium between on and off scHMM conformations. The observed rate constant for mant-ADP dissociation from scHMM is calcium dependent, and we show here that it can be used to define the equilibrium constant (K(eq)) between on and off conformations. The data show that K(eq) is markedly ionic strength dependent, with high salt (>/=200 mM) abolishing the off state even in the absence of calcium and low salt (<50 mM) favoring the off state even in the presence of calcium. Debye-Huckel plots of the equilibrium constant (K(eq)) for the on and off forms gave parallel slopes (5.94 +/- 0.33 and 6.36 +/- 0.17 M(-0.5)) in the presence and absence of calcium. The presence of an equilibrium mixture of two conformations was confirmed by sedimentation data and the effects of ADP, calcium and ionic strength were in qualitative agreement. Thus scHMM exists in two conformations that can be distinguished in sedimentation profiles and by the rate of release of mant-ADP. Increasing salt concentrations biases the system toward the on state, suggesting a role for ionic interactions in stabilizing the off state.

Cooperative regulation of myosin-actin interactions by a continuous flexible chain I: actin-tropomyosin systems.

We present a model for cooperative myosin binding to the regulated actin filament, where tropomyosins are treated as a weakly-confined continuous flexible chain covering myosin binding sites. Thermal fluctuations in chain orientation are initially required for myosin binding, leaving kinked regions under which subsequent myosins may bind without further distortion of the chain. Statistical mechanics predicts the fraction of sites with bound myosin-S1 as a function of their affinities. Published S1 binding curves to regulated filaments with different tropomyosin isoforms are fitted by varying the binding constant, chain persistence length nu (in actin monomers), and chain kink energy A from a single bound S1. With skeletal tropomyosin, we find an S1 actin-binding constant of 2.2 x 10(7) M(-1), A = 1.6 k(B)T and nu = 2.7. Similar persistence lengths are found with yeast tropomyosin. Larger values are found for tropomyosin-troponin in the presence of calcium (nu = 3.7) and tropomyosins from smooth muscle and fibroblasts (nu = 4.5). The relationship of these results to structural information and the rigid-unit model of McKillop and Geeves is discussed.

Cooperative regulation of myosin-actin interactions by a continuous flexible chain II: actin-tropomyosin-troponin and regulation by calcium.

The model of myosin regulation by a continuous tropomyosin chain is generalized to a chain of tropomyosin-troponin units. Myosin binding to regulated actin is cooperative and initially inhibited by the chain as before. In the absence of calcium, myosin is further inhibited by the binding of troponin-I to actin, which through the whole of troponin pins the tropomyosin chain in a blocking position; myosin and TnI compete for actin and induce oppositely-directed chain kinks. The model predicts equilibrium binding curves for myosin-S1 and TnI as a function of their first-order affinities K(S1) and L(TI). Myosin is detached by the actin binding of TnI, but TnI is more efficiently detached by myosin when the kink size (typically nine to ten actin sites) spans the seven-site spacing between adjacent TnI molecules. An allosteric mechanism is used for coupling the detachment of TnI to calcium binding by TnC. With thermally activated TnI kinks (kink energy B approximately k(B)T), TnI also binds cooperatively to actin, producing cooperative detachment of myosin and biphasic myosin-calcium Hill plots, with Hill coefficients of 2 at high calcium and 4-6 at low calcium as observed in striated muscle. The theory also predicts the cooperative effects observed in the calcium loading of TnC.

Differing ADP release rates from myosin heavy chain isoforms define the shortening velocity of skeletal muscle fibers.

To understand mammalian skeletal myosin isoform diversity, pure myosin isoforms of the four major skeletal muscle myosin types (myosin heavy chains I, IIA, IIX, and IIB) were extracted from single rat muscle fibers. The extracted myosin (1-2 microg/15-mm length) was sufficient to define the actomyosin dissociation reaction in flash photolysis using caged-ATP (Weiss, S., Chizhov, I., and Geeves, M. A. (2000) J. Muscle Res. Cell Motil. 21, 423-432). The ADP inhibition of the dissociation reaction was also studied to give the ADP affinity for actomyosin (K(AD)). The apparent second order rate constant of actomyosin dissociation gets faster (K(1)k(+2) = 0.17 -0.26 microm(-1) x s(-1)), whereas the affinity for ADP is weakened (250-930 microm) in the isoform order I, IIA, IIX, IIB. Both sets of values correlate well with the measured maximum shortening velocity (V(0)) of the parent fibers. If the value of K(AD) is controlled largely by the rate constant of ADP release (k(-AD)), then the estimated value of k(-AD) is sufficiently low to limit V(0). In contrast, [ATP]K(1)k(+2) at a physiological concentration of 5 mm ATP would be 2.5-6 times faster than k(-AD).

Kinetic resolution of a conformational transition and the ATP hydrolysis step using relaxation methods with a Dictyostelium myosin II mutant containing a single tryptophan residue.

The fluorescence emission intensity from a conserved tryptophan residue (W501) located in the relay loop (F466 to L516) of the Dicytostelium discoideum myosin II motor domain is sensitive to ATP binding and hydrolysis. The initial binding process is accompanied by a small quench in fluorescence, and this is followed by a large enhancement that appears coincident with the hydrolysis step. Using temperature and pressure jump methods, we show that the enhancement process is kinetically distinct from but coupled to the hydrolysis step. The fluorescence enhancement corresponds to the open-closed transition (k(obs) approximately 1000 s(-1) at 20 degrees C). From the overall steady-state fluorescence signal and the presence or absence of a relaxation transient, we conclude that the ADP state is largely in the open state, while the ADP.AlF(4) state is largely closed. At 20 degrees C the open-closed equilibria for the AMP.PNP and ADP.BeF(x) complexes are close to unity and are readily perturbed by temperature and pressure. In the case of ATP, the equilibrium of this step slightly favors the open state, but coupling to the subsequent hydrolysis step gives rise to a predominantly closed state in the steady state. Pressure jump during steady-state ATP turnover reveals the distinct transients for the rapid open-closed transition and the slower hydrolysis step.

KIF1D is a fast non-processive kinesin that demonstrates novel K-loop-dependent mechanochemistry.

The KIF1 subfamily members are monomeric and contain a number of amino acid inserts in surface loops. A particularly striking insertion of several lysine/arginine residues occurs in L12 and is called the K-loop. Two recent studies have employed both kinetic and single-molecule methods to investigate KIF1 motor properties and have produced very different conclusions about how these motors generate motility. Here we show that a hitherto unstudied member of this group, KIF1D, is not chemically processive and drives fast motility despite demonstrating a slow ATPase. The K-loop of KIF1D was analysed by deletion and insertion mutagenesis coupled with characterization by steady state and transient kinetics. Together, the results indicate that the K-loop not only increases the affinity of the motor for the MT, but crucially also inhibits its subsequent isomerization from weak to strong binding, with coupled ADP release. By stabilizing the weak binding, the K-loop establishes a pool of motors primed to undergo their power stroke.

Regulatory properties of tropomyosin effects of length, isoform, and N-terminal sequence.

The regulatory properties of naturally occurring tropomyosins (Tms) of differing lengths have been examined. These Tms span from 4 to 7 actin subunits. Native proteins have been used to study the common 7 actin-spanning skeletal and smooth muscle variants and expressed recombinant proteins to study the shorter fibroblast 5a, 5b, yeast Tm1 and yeast Tm2 Tms (6, 6, 5, and 4 actin-spanning variants, respectively). The yTm2 has been overexpressed in Escherichia coli with N-terminal constructs equivalent to those previously used for yTm1 [Maytum, R., et al. (2000) Biochemistry 39, 11913]. The regulation of myosin subfragment 1 (S1) binding to actin by Tm has been assessed using a sensitive S1 binding titration. The equilibrium between closed and open (C to M states, KT = 0.1-0.14) was similar for all vertebrate Tms. Apart from skTm where the apparent cooperative unit size (n) is the same as the structural size (n = 7 actin sites), the other vertebrate Tms that were studied exhibited large n values (n = 12-14). The yeast Tms also exhibited large values of n (6-9) in comparison to their structural sizes (4-5). The determined value of KT depended on the N-terminal sequence (KT = 0.15-1). These results are compared with the effect of S1 upon Tm's affinity for actin. The yeast Tms have regulatory parameters similar to those of skTm, but unlike skTm, S1 has little effect upon their actin affinity. This shows that an actin state with a high affinity for S1 and Tm is not necessary for regulation, and the higher affinity of S1 for actin in the presence of vertebrate Tms is probably the result of a direct interaction of S1 with Tm.

ADP binding induces an asymmetry between the heads of unphosphorylated myosin.

Light chain phosphorylation is the key event that regulates smooth and non-muscle myosin II ATPase activity. Here we show that both heads of smooth muscle heavy meromyosin (HMM) bind tightly to actin in the absence of nucleotide, irrespective of the state of light chain phosphorylation. In striking contrast, only one of the two heads of unphosphorylated HMM binds to actin in the presence of ADP, and the heads have different affinities for ADP. This asymmetry suggests that phosphorylation alters the mechanical coupling between the heads of HMM. A model that incorporates strain between the two heads is proposed to explain the data, which have implications for how one head of a motor protein can gate the response of the other.

Overexpression of human cardiac troponin in Escherichia coli: its purification and characterization.

All three subunits of the human cardiac troponin complex (cTn), namely the major isoform of the tropomyosin binding subunit (hcTnT3), the inhibitory subunit (cTnI), and the calcium binding subunit (cTnC), have been coexpressed in Escherichia coli. The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E. coli. The coexpressed subunits assembled within the bacterial cells to form the hcTn complex (hcTnT3.hcTnI.hcTnC). The complex was isolated and purified by three chromatographic steps. Per 6-L cell culture about 10 mg of a highly purified troponin complex showing the expected 1:1:1 molar ratio of hcTnT3:cTnI:cTnC was obtained. Upon phosphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombinant troponin complex shows a nearly identical (31)P NMR spectrum to the native one isolated from bovine heart. By measuring the rate of myosin S1 binding to reconstituted thin filaments it was shown that the dependence of the regulation of S1 binding upon calcium concentration and bisphosphorylation was comparable to the native complex.

A flash photolysis fluorescence/light scattering apparatus for use with sub microgram quantities of muscle proteins.

Transient kinetic methods such as stopped flow and quenched flow have been used to elucidate many of the fundamental features of the molecular interactions which underlie muscle contraction. However, these methods traditionally require relatively large amounts of protein (10(-3) g) and so have been used most effectively for the proteins purified from bulk muscle tissue of large animals or where the proteins can be expressed in large amounts (e.g.. Dictyostelium). We have investigated the use of flash photolysis of an inert precursor of ATP (cATP) to initiate the dissociation of acto.S1 and acto.myosin and the subsequent ATP turnover reaction. Using a sample volume of 10 microl we show that a significant amount of information on the transient and steady-state kinetics of the system can be obtained from a sample containing just 50 nM of acto.myosin or acto.S1 complex in solution. Therefore in presence of excess of one protein component the measurements require only 250 ng myosin, 62 ng S1 or 25 ng actin. This is therefore the method of choice for kinetic analysis of acto.myosins which are only available in microgram quantities. We report for the first time the determination of the second order rate constant of ATP-induced dissociation of actin from the myosin extracted from a single fibre from a rabbit psoas muscle.

Actomyosin regulatory properties of yeast tropomyosin are dependent upon N-terminal modification.

The yeast tropomyosin 1 gene (TPM1) encodes the major isoform of the two tropomyosins (Tm) found in yeast. The gene has been expressed in E. coli and the protein purified. The gene product (yTm1) is a 199-amino acid protein that has a low affinity for actin compared to the native yTm1 purified from yeast. Mass spectrometry shows that the native protein is acetylated while the recombinant protein is not. A series of yTm1 N-terminal constructs were made with either an Ala-Ser dipeptide extension previously shown to restore actin binding to skeletal muscle Tm or the natural extension found in fibroblast Tm 5a/b. All constructs bound actin tightly and showed similar CD spectra and thermal stability. All constructs induced cooperativity in the equilibrium binding of myosin subfragment 1, to actin but the binding curves differed significantly between the constructs. The apparent cooperative unit size (n) and closed/open equilibrium (K(T)) were determined using a fluorescence titration technique [Maytum et al. (1998) Biophys. J. 74, A347]. The data could be accounted for by changes in K(T) (0.1-1) with no change in n. Values of n were approximately twice the structural unit size (5 actin sites). The presence of yTm on actin had little effect upon the overall affinity of S1 for actin despite showing an ability to regulate the acto-myosin interaction. These results show that the short yTm can aid our understanding of actomyosin regulation and that the N-terminus of Tm has a major influence upon its regulatory properties.

Stabilization of the actomyosin complex by negative charges on myosin.

Sequence comparisons of members of the myosin superfamily show a high degree of charge conservation in a surface exposed helix (Dictyostelium discoideum myosin II heavy chain residues S510 to K546). Most myosins display a triplet of acidic residues at the equivalent positions to D. discoideummyosin II residues D530, E531, and Q532. The high degree of charge conservation suggests strong evolutionary constrain and that this region is important for myosin function. Mutations at position E531 were shown to strongly affect actin binding [Giese, K. C., and Spudich, J. A. (1997) Biochemistry 36, 8465-8473]. Here, we used steady-state and transient kinetics to characterize the enzymatic competence of mutant constructs E531Q and Q532E, and their properties were compared with those of a loop 2 mutant with a 20 amino acid insertion containing 12 positive charges (20/+12) [Furch et al. (1998) Biochemistry 37, 6317-6326], double mutant Q532E(20/+12), and the native motor domain constructs. Our results confirm that charge changes at residues 531 and 532 primarily affect actin binding with little change being communicated to the nucleotide pocket. Mutation D531Q reduces actin affinity (K(A)) 10-fold, while Q532E leads to a 5-fold increase. The observed changes in K(A)() stem almost exclusively from variations in the dissociation rate constant (k(-A)), with the introduction of a single negative charge at position 532 having the same effect on k(-A) as the introduction of 12 positive charges in the loop 2 region.

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments.

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.