James S. Evans

Proton magnetic resonance studies on proteolytic fragments of troponin-C. Structural homology with the native molecule

Comparison of proton magnetic resonance spectra of a tryptic and a thrombin fragment of troponin-C with that of the native protein has identified the domain of the molecule influenced by Ca2+ binding to the lower affinity regions I and II of troponin-C. The binding of Ca2+ to these sites results in a subtle alteration of the tertiary fold of the N-terminal half of troponin-C involving weakened contacts between several hydrophobic groups. The role and kinetics of the movements within the troponin-C molecule associated with binding at the regulatory sites are discussed.

HERPESVIRAL THYMIDINE KINASES : LAXITY AND RESISTANCE BY DESIGN

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Phosphorylation of the minimal inhibitory region at the C-terminus of caldesmon alters its structural and actin binding properties

Caldesmon is an inhibitory protein believed to be involved in the regulation of thin filament activity in smooth muscles and is a major cytoplasmic substrate for MAP kinase. NMR spectroscopy shows that the actin binding properties of the minimal inhibitory region of caldesmon, residues 750-779, alter upon MAP kinase phosphorylation of Ser-759, a residue not involved in actin binding. This phosphorylation leads to markedly diminished actin affinity as a result of the loss of interaction at one of the two sites that bind to F-actin. The structural basis for the altered interaction is identified from the observation that phosphorylation destabilises a turn segment linking the two actin binding sites and thereby results in the randomisation of their relative disposition. This modulatory influence of Ser-759 phosphorylation is not merely a function of the bulkiness of the covalent modification since the stability of the turn region is observed to be sensitive to the ionisation state of the phosphate group. The data are discussed in the context of the inhibitory association of the C-terminal domain of caldesmon with F-actin.

The Regulatory Effects of Tropomyosin and Troponin-I on the Interaction of Myosin Loop Regions with F-actin

The N terminus of skeletal myosin light chain 1 and the cardiomyopathy loop of human cardiac myosin have been shown previously to bind to actin in the presence and absence of tropomyosin (Patchell, V. B., Gallon, C. E., Hodgkin, M. A., Fattoum, A., Perry, S. V., and Levine, B. A. (2002) Eur. J. Biochem. 269, 5088–5100). We have extended this work and have shown that segments corresponding to other regions of human cardiac β-myosin, presumed to be sites of interaction with F-actin (residues 554–584, 622–646, and 633–660), likewise bind independently to actin under similar conditions. The binding to F-actin of a peptide spanning the minimal inhibitory segment of human cardiac troponin I (residues 134–147) resulted in the dissociation from F-actin of all the myosin peptides bound to it either individually or in combination. Troponin C neutralized the effect of the inhibitory peptide on the binding of the myosin peptides to F-actin. We conclude that the binding of the inhibitory region of troponin I to actin, which occurs during relaxation in muscle when the calcium concentration is low, imposes conformational changes that are propagated to different locations on the surface of actin. We suggest that the role of tropomyosin is to facilitate the transmission of structural changes along the F-actin filament so that the monomers within a structural unit are able to interact with myosin.

Protein-protein interaction sites in the calcium modulated skeletal muscle troponin complex.

The sequence domains that contribute to the surfaces of contact between Troponin-C and the other regulatory protein subunits of skeletal muscle troponin are proposed on the basis of data obtained by proton magnetic resonance and other physicochemical studies on the interaction with Troponin-I of both Troponin-C and its peptide fragments. Marked sequence homology in Troponin-C from various species is found for the residues involved in subunit linkage. The role of the recognition sites is discussed.

Proton magnetic resonance studies on peptide fragments of troponin-C containing single calcium-binding sites

Proton magnetic resonance spectroscopy has been employed to study the solution conformation of three cleavage fragments of troponin-C, each containing a single Ca(II)-binding site and corresponding to different regions in the primary sequence; viz. CB8 (residues 46-77), CB9 (residues 85-134) and TH2 (residues 121-159). Although all three peptides lack a well-defined tertiary fold in the absence of metal ions, several spectral features indicate the presence of local conformational constraints in each apo-peptide. Ca(II) binding led to spectral changes consistent with increased restriction of backbone motility and the adoption of a more compact conformation. Studies using paramagnetic ions as conformational probes support current views concerning the nature of the ligands at the metal binding sites. The nature and kinetics of the structural influence of metal binding suggest that the conformational constraints existing in the CB8 apo-peptide provide an adequate Ca(II)-binding configuration. In contrast, the CB9 and TH2 peptides exhibit spectral changes consistent with an increased local structure in the region of helix E (residues 94-102) in the case of CB9 and helix H (residues 148-159) in the case of TH2. In CB9, conformation changes also appear to be transmitted to a portion of the sequence (residues 87-93) preceding helix E, a putative site of interaction between troponin-C and troponin-I. These data are discussed with reference to the contribution of long-range (interdomain) interactions within troponin-C and the modulation of troponin subunit protein-protein interactions by Ca(II) binding.