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Results demonstrate that phosphorylation of the cardiac-specific N-terminus of troponin I reduces the binding affinity of cardiac troponon C for cardiac troponin I. Data suggest that beta-adrenergic modulation of troponin sensitivity for calcium occurs via an interaction between the cardiac-specific N-terminus troponin I and troponin C regularotry domain.

Results show that dolichyl-phosphate-mannose synthase synthesizes dolichol-phosphate mannose, is associated with endoplasmic reticulum, and requires lipid for its activity. Investigations on the active site of the enzyme reveals that consensus sequence is not necessary for substrate recognition or for the catalytic activity.

A study was conducted on the interaction between cTnI and cTnC fragments, cTnC1-89 and cTnC90-162, in an effort to understand how the phosphorylation signal at one end of the complex affects the Ca2+ binding site at the other end. The findings indicate that the desensitization of Ca2+ binding to the regulatory domain of cTnC can take place even in the absence of cTnC90-162 or the central helix of cTnC. This further suggests that phosphorylation-induced changes are trasmitted to the regulatory site of cTnC through a global change in cTnI structure.

The effect of phosphorylation on the conformation of the N-terminal extension of cardiac cTnI was established through utilization of time-resolved fluorescence measurements. Fluorescence intensity decay of IAANS attached to Cys-5 of cTnI mutant, cTnI(S5C/C81I/C98S), revealed that phosphorylation yielded a change in the shift of the emission spectrum, decreased the weighted mean lifetime, and decreased correlation time.

The effect of phosphorylation on the distance between the sites located at the C-terminus and N-terminus of the (cTnI) was determined through utilization of fluorescence resonance energy transfer (FRET). Results have shown that both the mean distance and the half-width of the distribution were significantly reduced following PKA-induced phosphorylation of cTnI. It was shown that the phosphorylation induced the folding of the N-terminal extension towards the C-terminal region resulting to altered catalytic activity.

The intersite distances from Cys374 of actin to Cys707 (SH1) and Cys697 (SH2) of myosin subfragment 1 (S1) in actosubfragment 1 through fluorescence resonance energy transfer for rigor complexes having bound ADP and ADP plus orthovandate, A-S1-ADP, and A-S1-ADP-Vi are calculated. The findings show that in the steady-state hydrolysis of ATP, or in the weak-binding condition of actomyosin, the short helical portion of S1 heavy chain containing SH1 comes nearer to the COOH-terminal end of actin. But the neighboring helical portion having SH2 stays stable.

Use of troponin C (CTnC), with changes in Cys84 and Cys35 residues, and a fluorescent probe to examine the interactions between CTnC and CTnI in bovine cardiac muscle reveals that the interactions lead to calcium activation and a coupling free energy of minus 0.65 Kcal. 3', 5'-cAMP-dependent protein kinase-induced phosphorylation of CTnI causes a 2.5 to 3.5 fold reduction in the association constants, resulting in the loss of sensitivity of calcium. Phosphatase treatment to the phosphorylated CTnI helps reinstate calcium sensitivity.

The intensity and anisotrophy decays of the single tryptophan in bovine brain S-100a (alpha, beta) protein were determined by time-resolved fluorescence spectroscopy. The effects of Ca2+-binding on the conformation of the protein was studied at pH 7. 2 and 8.4. The results show that the binding of calcium ion induces a conformational change in the protein and indicate differences in the localized and global conformations of the protein at the two pH values.

The effects of bound regulatory [Ca.sup.2+], strongly bound myosin, and PKA bisphosphorylation of cardiac troponin 1 (cTn1) on the distance changes from actin Cys374 to several actin-binding sites within the inhibitory region, regulatory region, and the second actin-binding region of cTn1 are studied. The results provide quantitative information on a two-step movement of the inhibitory/regulatory region of cTn1 residues 130-167 from actin filament following [Ca.sup.2+] activation and strong cross-bridge formation between myosin S1 and actin.

Experiments are conducted to compare the tension and thin filament Ca(super 2+) signaling in preparations containing either wild-type cardiac troponin I or a mutant cTnI, with an R146G mutation [cTnI(146G)] linked to familial hypertrophic cardiomyopathy. The result demonstrates that the activation of cardiac myofilaments is tightly coupled to the open state of the N-domain of cTnC, and supports the hypothesis that pathological effects of phosphorylation are influenced by mutations in cTnI.

The three calcium-saturated subunits of cardiac troponin exhibit marked conformational changes and reorientations in response to phosphorylation of cTnI component. The organization of the subunits within the unphosphorylated complex is probed using deuterated cTnI, which shows the shape of the cTnI component within the ternary complex.

Research points out that the optical transduction sensing in the skeletal myosin is induced by hypochronism in the ATP-sensitive tryptophan absorption band. Crystallographic analysis of the myosin structures indicates that ATP-sensitive tryptophan hypochromism is less than 10% of the experimetnally determined value, suggesting a need for determining structural manifestation of optical transduction sensing in myosin.

Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin II. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to an identifiable state change. These studies indicate that for this myosin:ADP, both the catalytic site and the actin binding site can each assume one of two conformations. Relatively loose coupling between these two binding sites leads to three discrete actin-associated ADP states. After an initial, weakly bound state, binding of myosin:ADP to actin shifts the equilibrium toward a mixture of two states that each bind actin strongly, but differ in the conformation of their catalytic sites. By contrast, fast myosins, including Dictyostelium myosin II, have reciprocal coupling between the actin and ADP binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly bound actomyosin:ADP state in smooth muscle, would prolong the fraction of the ATPase cycle time that this actomyosin spends in a force-generating conformation, and may be central to explaining the physiologic differences between this and other myosins.