Myosin-binding protein C (MyBP-C) is a multidomain protein present in the thick filaments of striated muscles and is involved in both sarcomere formation and contraction regulation. The latter function is believed to be located at the N terminus, which is close to the motor domain of myosin. The cardiac isoform of MyBP-C is linked to hypertrophic cardiomyopathy. Here, we use NMR spectroscopy and biophysical and biochemical assays to study the three-dimensional structure and interactions of the cardiac-specific Ig-like domain C0, a part of cardiac MyBP-C of which little is known. The structure confirmed that C0 is a member of the IgI class of proteins, showing many of the characteristic features of this fold. Moreover, we identify a novel interaction between C0 and the regulatory light chain of myosin, thus placing the N terminus of the protein in proximity to the motor domain of myosin. This novel interaction is disrupted by several cardiomyopathy-linked mutations in the MYBPC3 gene. These results provide new insights into how cardiac MyBP-C incorporates in the sarcomere and how it can contribute to the regulation of muscle contraction.

A novel Lanthanide Induced Shift Analysis (LISA) is presented. In this analysis both the paramagnetic and the diamagnetic lanthanide induced chemical shifts are normalised separately in contrast to previous techniques in which only the paramagnetic shifts were normalised. This procedure is used together with molecular mechanics (MMFF94) and ab initio (RHF/6-31G, RHF/6-311G Ãà and B3LYP/6-311G Ãà ) calculations to investigate s-cis/s-trans isomerism in some a,b-unsaturated aldehydes, ketones and esters. In tiglic aldehyde 1 and trans-cinnamaldehyde 4 the s-trans conformer predominates with energy differences DE (s-cis-s-trans) of 1.64 and 1.76 kcal/mol. In methyl vinyl ketone 2 and trans-cinnamyl methyl ketone 5 the populations of the s-cis and s-trans isomers are almost equal (DE 0.24 and 0.0 kcal/mol) and in methyl crotonate 3 and methyl trans-cinnamate 6 the s-cis conformer is more stable (DE À0.72 and À0.41 kcal/mol). These results are in agreement with both the MMFF94 and ab initio calculated energies for the compounds except tiglic aldehyde 1 in which all the calculated values are too large and cinnamyl ketone 5. In this compound the ab initio calculations predict the s-cis form to be more stable than the s-trans in contrast to both the MM calculations and the observed result which give both forms of equal energy. Also in both the MM and ab initio calculations phenyl substitution in the ketone (2 vs. 5) considerably stabilises the s-cis form. This is not observed in practise. In phenyl acetate 7 the B3LYP calculations give two equally stable structures, one planar one non-planar. The MMFF94 and MP2 calculations and the LIS analysis support the existence of only the non-planar conformer in solution, which is also the conformation of phenyl acetate in the crystal. CONFORMATIONAL ANALYSIS OF a,b-UNSATURATED CARBONYL COMPOUNDS 385 a [S] 0 0.5 Ml À1 b Yb(fod) 3 experiment: r  10 À2 0.00, 1.87, 4.31, 9.64, corresponding coefficients > 0.9993. c Lu(fod) 3 experiment: r  10 À2 0.00, 4.02, 7.13, 10.49, corresponding coefficients > 0.9988. d Yb(fod) 3 experiment: r  10 À2 0.00, 4.39, 7.49, 13.74, corresponding coefficients > 0.9993. e Lu(fod) 3 experiment: r  10 À2 0.00, 3.79, 5.48, 8.88, corresponding coefficients > 0.9950. f Yb(fod) 3 experiment: r  10 À2 0.00, 2.65, 5.45, 8.13, 11.00, corresponding coefficients > 0.9998. g Lu(fod) 3 experiment: r  10 À2 0.00, 1.37, 2.72, 4.20, 5.72, corresponding coefficients > 0.9989.

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