Darl R. Swartz

Effect of Calcium-sensitizing Mutations on Calcium Binding and Exchange with Troponin C in Increasingly Complex Biochemical Systems

The calcium-dependent interactions between troponin C (TnC) and other thin and thick filament proteins play a key role in the regulation of cardiac muscle contraction. Five hydrophobic residues (Phe(20), Val(44), Met(45), Leu(48), and Met(81)) in the regulatory domain of TnC were individually substituted with polar Gln, to examine the effect of these mutations that sensitized isolated TnC to calcium on (1) the calcium binding and exchange with TnC in increasingly complex biochemical systems and (2) the calcium sensitivity of actomyosin ATPase. The hydrophobic residue mutations drastically affected calcium binding and exchange with TnC in increasingly complex biochemical systems, indicating that side chain intra- and intermolecular interactions of these residues play a crucial role in determining how TnC responds to calcium. However, the mutations that sensitized isolated TnC to calcium did not necessarily increase the calcium sensitivity of the troponin (Tn) complex or reconstituted thin filaments with or without myosin S1. Furthermore, the calcium sensitivity of reconstituted thin filaments (in the absence of myosin S1) was a better predictor of the calcium dependence of actomyosin ATPase activity than that of TnC or the Tn complex. Thus, both the intrinsic properties of TnC and its interactions with the other contractile proteins play a crucial role in modulating the binding of calcium to TnC in increasingly complex biochemical systems.

Myosin heavy chain isoform composition influences the susceptibility of actin-activated S1 ATPase and myofibrillar ATPase to pH inactivation

The objectives of this study were to determine the influence of pH and MyHC isoforms on myofibrillar and actin-activated myosin subfragment 1 (S1) ATPase activity and the protective effect of actin. Red (RST) semitendinosus and white (WST) semitendinosus myofibrils were incubated at pH 7, 6, or 5.5 with 0 or 2mM ATP. RST and WST S1 isolates were incubated at pH 7, 6, or 5.5 in the presence or absence of actin. Maximum calcium-activated myofibrillar and actin-activated S1-ATPase activity were then assayed at pH 7. Incubation of myofibrils with ATP caused ATPase activity of myofibrils to decrease (p<0.05) with the pH of the incubation. RST myofibrils maintained a higher (p<0.0001) relative activity than WST myofibrils after incubation at pH 6 with ATP. Myofibrils incubated without ATP exhibited higher (p<0.001) activities than those incubated with ATP following pH 5.5 treatments. WST myofibrils had a lower (p<0.05) relative activity than RST following incubation at pH 5.5 without ATP. S1 ATPase activities decreased (p<0.05) with incubation pH in WST samples, but not in RST samples. WST S1 activity was higher (p<0.01) in samples exposed to pH 6 and 5.5 with actin bound compared to those incubated without actin. RST S1 exhibited a higher (p<0.01) relative activity than WST samples following pH 5.5 treatment with bound actin. These data show that low pH inactivates myofibrils by altering actin-activated S1 ATPase. Furthermore, these results suggest that muscles with high proportions of fast fibers are more susceptible to pH inactivation of ATPase activity and that the protective effect of actin binding to myosin is less in fast fibers.

Fast Pressure Jumps Can Perturb Calcium and Magnesium Binding to Troponin C F29W

We have used rapid pressure jump and stopped-flow fluorometry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL/mol). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000/s and 100/s. Between pCa 8-5.4 and at troponin C concentrations of 8-28 muM, the slow relaxation times were invariant, indicating that a protein isomerization was rate-limiting. The fast event was only detected over a very narrow pCa range (5.6-5.4). We have devised a model based on a Monod-Wyman-Changeux cooperative mechanism with volume changes of -9 and +6 mL/mol for the calcium binding to the regulatory sites and closed to open protein isomerization steps, respectively. In the absence of magnesium, we discovered that calcium binding to the C-terminal sites could be detected, despite their position distal to the calcium-sensitive tryptophan, with a volume change of +25 mL/mol. We used this novel observation to measure competitive magnesium binding to the C-terminal sites and deduced an affinity in the range 200-300 muM (and a volume change of +35 mL/mol). This affinity is an order of magnitude tighter than equilibrium fluorescence data suggest based on a model of direct competitive binding. Magnesium thus indirectly modulates binding to the N-terminal sites, which may act as a fine-tuning mechanism in vivo.

Influence of salt and pyrophosphate on bovine fast and slow myosin S1 dissociation from actin.

The kinetics of myosin dissociation from actin was investigated and also the impact of salt, MgPPi, and myosin heavy chain isoform on myosin subfragment 1 (S1) dissociation from actin using purified proteins and fluorescence spectroscopy. Both NaCl and MgPPi increased myosin S1 dissociation rate. When salt concentrations increased from 0.1 to 1.0 M, the dissociation rate of S1 from bovine masseter (slow) and cutaneous trunci (fast) muscle increased 38 and 78 fold, respectively. MgPPi had an even greater effect on S1 dissociation from actin. With the addition of MgPPi to the mixture of pyrene actin and S1, the fluorescence increased about 85% within the dead time of the mixing approach.. Unlike salt, MgPPi had no apparent difference in its ability to dissociate slow or fast S1 isoforms from actin. The results reveal that salt and MgPPi increase myosin extraction and functionality in meat by weakening the actomyosin interaction and that some of the difference in the functionality of red and white muscle may be related to actomyosin dissociation.

Tripolyphosphate hydrolysis by bovine fast and slow myosin subfragment 1 isoforms.

Polyphosphates are used in the meat industry to increase the water holding capacity of meat products. Tripolyphosphate (TPP) is a commonly used polyphosphate and it is metabolized into pyrophosphate and monophosphate in meat. The enzymes responsible for its metabolism have not been fully characterized. The motor domain of myosin (subfragment 1 or S1) is a likely candidate. The objectives of this study were to determine if bovine S1 hydrolyzes TPP, to characterize the TPPase activity of the fast (cutaneous trunci) and slow (masseter) isoforms, and to determine the influence of pH on S1 TPPase activity. S1 hydrolyzed TPP and in comparison with ATP as substrate, it hydrolyzed TPP 16-32% more slowly. Fast S1 hydrolyzed both substrates faster compared to slow S1 and the difference between the isoforms was greater with TPP as the substrate. The V(max) was 0.94 and 5.0 nmol Pi/mg S1 protein/min while the K(m) was 0.38 and 0.90 mM TPP for slow and fast S1, respectively. Pyrophosphate was a strong inhibitor of TPPase activity with a K(i) of 88 and 8.3 microM PPi for fast and slow S1 isoforms, respectively. Both ATPase and TPPase activities were influenced by pH with the activity being higher at low pH for both fast and slow S1 isoforms. The activity at pH 5.4 was 1.5 to 4-fold higher than that at pH 7.6 for the different isoforms and substrates. These data show that myosin S1 readily hydrolyzes TPP and suggest that it is a major TPPase in meat.

Differences between Cardiac and Skeletal Troponin Interaction with the Thin Filament Probed by Troponin Exchange in Skeletal Myofibrils

Troponin (Tn) is the calcium-sensing protein of the thin filament. Although cardiac troponin (cTn) and skeletal troponin (sTn) accomplish the same function, their subunit interactions within Tn and with actin-tropomyosin are different. To further characterize these differences, myofibril ATPase activity as a function of pCa and labeled Tn exchange in rigor myofibrils was used to estimate Tn dissociation rates from the nonoverlap and overlap region as a function of pCa. Measurement of ATPase activity showed that skeletal myofibrils containing >96% cTn had a higher pCa 9 ATPase activity than, but similar pCa 4 activity to, sTn-containing myofibrils. Analysis of the pCa-ATPase activity relation showed that cTn myofibrils were more calcium sensitive but less cooperative (pCa50 = 6.14, nH = 1.46) than sTn myofibrils (pCa50= 5.90, nH = 3.36). The time course of labeled Tn exchange at pCa 9 and 4 were quite different between cTn and sTn. The apparent cTn dissociation rates were approximately 2-10-fold faster than sTn under all the conditions studied. The apparent dissociation rates for cTn were 5 x 10(-3) min(-1), 150 x 10(-3) min(-1), and 260 x 10(-3) min(-1), whereas for sTn they were 0.6 x 10(-3) min(-1), 88 x 10(-3) min(-1), and 68 x 10(-3) min(-1) for the nonoverlap region at pCa 9, nonoverlap region at pCa 4, and overlap region at pCa 4, respectively. Normalization of the apparent dissociation rates gives 1:30:50 for cTn compared with 1:150:110 for sTn (nonoverlap at pCa 9:nonoverlap at pCa 4:overlap at pCa 4) suggesting that calcium has a smaller influence, whereas strong cross-bridges have a larger influence on cTn dissociation compared with sTn. The higher cTn dissociation rate in the nonoverlap region and ATPase activity at pCa 9 suggest that it gives a less off or inactive thin filament. Analysis of the intensity ratio (after a short time of exchange) as a function of pCa showed that cTn had greater calcium sensitivity but lower cooperativity than sTn. In addition, the magnitude of the change in intensity ratio going from pCa 9 to 4 was less for cTn than sTn. These data suggest that the influence of calcium on cTn exchange is less than sTn even though calcium can activate ATPase activity to a similar extent in cTn compared with sTn myofibrils. This may be explained partially by cTn being less off or inactive at pCa 9. Modeling of the intensity profiles obtained after Tn exchange at pCa 5.8 suggest that the profiles are best explained by a model that includes a long-range cross-bridge effect that grades with distance from the rigor cross-bridge for both cTn and sTn.

A kinetic model of troponin dissociation in relation to thin filament regulation in striated muscle

The apparent rate of troponin (Tn) dissociation from myofibrils has been used as a method to study thin filament regulation in striated muscle. The rate is dependent upon calcium and strong crossbridges and supports the three-state model for thin filament regulation. The dissociation rate of Tn is extremely low so it is not intuitively clear that such a slow process would probe thin filament regulation. We have investigated this issue by developing a simple kinetic model to explain the Tn dissociation rate measured by labeled Tn exchange in the myofibrils. Tn is composed of three interacting subunits, TnC, TnI and TnT. In our model, TnIs regulatory domain switches from actin-tropomyosin to TnC followed by TnT dissociation from actin-tropomyosin. This TnI regulatory domain switching is linked to the transition of the thin filament from the blocked state to the closed state. It is calcium dependent and several orders of magnitude faster than TnT dissociation from actin-tropomyosin. By integrating the dimensionless rate equations of this model, we have computed the time course of each of the various components. In our numerical simulations, the rate constant for TnI switching from actin-tropomyosin to TnC was varied from 10 s⁻¹ to 1000 s⁻¹ to simulate the low calcium, blocked state to high calcium, closed state. The computed progress curves for labeled Tn exchange into the myofibrils and the derived intensity ratio between the non-overlap and overlap regions well explains the intensity ratio progress curves observed experimentally. These numerical simulations and experimental observations reveal that the apparent rate of Tn dissociation probes the blocked state to closed state equilibrium of the myofibrillar thin filament.

Ionic Strength Influence on Myofibril Thin Filament State Probed by Troponin Exchange

Activation of striated muscle contraction is a cooperative process initiated by calcium binding to troponin C (TnC). This is a solid-phase signal transduction process involving 3 states of thin filament: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interaction, and an fully activated open or M state. Here we evaluated the influence of ionic strength on Tn dissociation in relationship to myofibril thin filament state. The data we obtained showed that ionic strength had a significant influence on Tn dissociation with low ionic strength increasing Tn dissociation rate from non-overlap region (B or C state) and high ionic strength increasing Tn dissociation rate from overlap region (M state) of myofibril thin filament in both absence and presence of calcium. When ionic strength was decreased to ≤ 50 mM, Tn dissociation from non-overlap region became even faster than overlap region in the absence of calcium, suggesting that actin-tropomyosin in the non-overlap region actually changed from the inhibited B state to the C state at low ionic strength. Also, low ionic strength increased myofibril ATPase activity in the absence of calcium and the calcium-sensitivity of ATPase activity. Based on this and our current studies, we propose a refined kinetic scheme for Tn dissociation from myofibril thin filament, which suggests that the regulatory dissociation of TnI from actin-tropomyosin and association to TnC is not necessarily calcium coupled. Low ionic strength favors the B to C state equilibrium towards the C state (Head et al., Eur. J. Biochem, 227: 694) by favoring TnI dissociation from actin and possibly association with TnC in the absence of calcium. This well explains reported inconsistence between ATPase activity and S1 binding to actin under low ionic strength.