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Myosin binding protein C is a protein of the myosin filaments of striated muscle which is expressed in isoforms specific for cardiac and skeletal muscle. The cardiac isoform is phosphorylated rapidly upon adrenergic stimulation of myocardium by cAMP-dependent protein kinase, and together with the phosphorylation of troponin-I and phospholamban contributes to the positive inotropy that results from adrenergic stimulation of the heart. Cardiac myosin binding protein C is phosphorylated by cAMP-dependent protein kinase on three sites in a myosin binding protein C specific N-terminal domain which binds to myosin-S2. This interaction with myosin close to the motor domain is likely to mediate the regulatory function of the protein.Cardiac myosin binding protein C is a common target gene of familial hypertrophic cardiomyopathy and most mutations encode N-terminal subfragments of myosin binding protein C. The understanding of the signalling interactions of the N-terminal region is therefore important for understanding the pathophysiology of myosin binding protein C associated cardiomyopathy. We demonstrate here by cosedimentation assays and isothermal titration calorimetry that the myosin-S2 binding properties of the myosin binding protein C motif are abolished by cAMP-dependent protein kinase-mediated trisphosphorylation, decreasing the S2 affinity from a K d of W W5 W WM to undetectable levels. We show that the slow and fast skeletal muscle isoforms are no cAMP-dependent protein kinase substrates and that the S2 interaction of these myosin binding protein C isoforms is therefore constitutively on. The regulation of cardiac contractility by myosin binding protein C therefore appears to be a`brake-off' mechanism that will free a specific subset of myosin heads from sterical constraints imposed by the binding to the myosin binding protein C motif.z 1999 Federation of European Biochemical Societies.

Abstract-Myosin binding protein C (MyBP-C) is one of the major sarcomeric proteins involved in the pathophysiology of familial hypertrophic cardiomyopathy (FHC). The cardiac isoform is tris-phosphorylated by cAMP-dependent protein kinase (cAPK) on ␤-adrenergic stimulation at a conserved N-terminal domain (MyBP-C motif), suggesting a role in regulating positive inotropy mediated by cAPK. Recent data show that the MyBP-C motif binds to a conserved segment of sarcomeric myosin S2 in a phosphorylation-regulated way. Given that most MyBP-C mutations that cause FHC are predicted to result in N-terminal fragments of the protein, we investigated the specific effects of the MyBP-C motif on contractility and its modulation by cAPK phosphorylation. The diffusion of proteins into skinned fibers allows the investigation of effects of defined molecular regions of MyBP-C, because the endogenous MyBP-C is associated with few myosin heads. Furthermore, the effect of phosphorylation of cardiac MyBP-C can be studied in a defined unphosphorylated background in skeletal muscle fibers only. Triton skinned fibers were tested for maximal isometric force, Ca 2ϩ /force relation, rigor force, and stiffness in the absence and presence of the recombinant cardiac MyBP-C motif. The presence of unphosphorylated MyBP-C motif resulted in a significant (1) depression of Ca 2ϩ -activated maximal force with no effect on dynamic stiffness, (2) increase of the Ca 2ϩ sensitivity of active force (leftward shift of the Ca 2ϩ /force relation), (3) increase of maximal rigor force, and (4) an acceleration of rigor force and rigor stiffness development. Tris-phosphorylation of the MyBP-C motif by cAPK abolished these effects. This is the first demonstration that the S2 binding domain of MyBP-C is a modulator of contractility. The anchorage of the MyBP-C motif to the myosin filament is not needed for the observed effects, arguing that the mechanism of MyBP-C regulation is at least partly independent of a "tether," in agreement with a modulation of the head-tail mobility. Soluble fragments occurring in FHC, lacking the spatial specificity, might therefore lead to altered contraction regulation without affecting sarcomere structure directly. (Circ Res. 2000;86:51-58.)

Homing endonucleases are enzymes that catalyze the highly sequence-specific cleavage of DNA. We have developed an in vivo selection in Escherichia coli that links cell survival with homing endonuclease-mediated DNA cleavage activity and sequence specificity. Using this selection, wild-type and mutant variants of three homing endonucleases were characterized without requiring protein purification and in vitro analysis. This selection system may facilitate the study of sequence-specific DNA cleaving enzymes, and selections based on this work may enable the evolution of homing endonucleases with novel activities or specificities.

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The solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into protein crystals. Several 29-halogenated nucleotides have been reported as potential universal phasing tools for nucleotide binding proteins. However, only limited data are available dealing with the effect of 29-substitution on recognition by the protein. We have determined equilibrium dissociation constants of 29-halogenated ATP analogues for the ATP binding proteins UMP0CMP kinase and the molecular chaperone DnaK. Whereas the affinities to UMP0CMP kinase are of the same order of magnitude as for unsubstituted ATP, the affinities to DnaK are drastically decreased to undetectable levels. For 29-halogenated GTP analogues, the kinetics of interaction were determined for the small GTPases p21 ras~Y 32W! fluorescent mutant! and Rab5. The rates of association were found to be within about one order of magnitude of those for the nonsubstituted nucleotides, whereas the rates of dissociation were accelerated by factors of ;100~p21 ras ! or ;10 5~R ab5!, and the resulting equilibrium dissociation constants are in the nm or mM range, respectively. The data demonstrate that 29halo-ATP and -GTP are substrates or ligands for all proteins tested except the chaperone DnaK. Due to the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 10 5 -fold decrease in affinity as observed for Rab5 places the equilibrium dissociation constant in the mM range, so that they are still well suited for crystallization of the G-protein:nucleotide complex.Keywords: 29I0Br-ATP; 29I0Br-AppNHp; 29I0Br-GppNHp; fluorescence spectroscopy; phase problem; X-ray crystallography A large number of proteins interact with ATP or GTP. While the three-dimensional~3D! structures of many of these proteins are known, a large number remain to be determined. Despite homologies between many proteins and classes of proteins that interact with ATP or GTP, 3D structural homology is seldom high enough to allow structural determination from X-ray crystallographic data using molecular replacement only. Thus, the usual problem of determining the phases of the reflections remains, and the most commonly used method~multiple isomorphous replacement~MIR!! still involves the incorporation of heavy atoms into protein crystals. After crystallization, finding such derivatives is the second major bottle neck in the determination of the 3D structure of biomacromolecules. In recent years, a second method, that of multiple wavelength anomalous dispersion~MAD!, has become frequently used~Ogata, 1998!.Whereas most labeling procedures focus on the protein itself, only a few attempts have been made to incorporate foreign atoms into the ligands of proteins. In principle, advantage can be taken of the natural affinity of the ligand to the protein. Optimally, the modified ligand should fulfill three criteria:~1! The incorporated heavy! atom~s! should be suitable for the common phasing methods~MIR or MAD!;~2! the incorporation of the h...

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