CA2-ATOMS IN CALMODULIN AND OTHER EF-HAND PROTEINS - N-15 CHEMICAL-SHIFTS AS PROBES FOR MONITORING INDIVIDUAL-SITE CA2+ COORDINATION( COORDINATION TO BACKBONE CARBONYL OXYGEN)

Examination of the NMR N-15 chemical shifts of a number of EF-hand pro teins shows that the shift value for the amido nitrogen of the residue in position 8 of a canonical EF-hand loop (or position 10 of a pseudo EF-hand loop) provides a good indication of metal occupation of that site. The NH of the residue in position 8 is covalently bonded to the carbonyl of residue 7, the only backbone carbonyl that coordinates to the metal ion in a canonical EF-hand loop. Upon metal coordination to this carbonyl, there is an appreciable deshielding of the N-15 nucleus at position 8 (+4 to +8 ppm) due to the polarization of the O(7)=C(7) -N(8) amido group and the corresponding reduction in the electron dens ity of the nitrogen atom. This deshielding effect is effectively indep endent of the binding of metal to the other site of an EF-hand pair, a llowing the N-15 shifts to be used as probes for site-specific occupan cy of metal binding sites. In addition, a Ca2+-induced change in side- chain H-alpha-C-alpha-C-beta-H-beta torsion angle for isoleucine or va line residues in position 8 can also contribute to the deshielding of the amide N-15 nucleus. This conformational effect occurs only in site s I or III and takes place upon binding a Ca2+ ion to the other site o f an EF-hand pair (site II or IV) regardless of whether the first site is occupied. The magnitude of this effect is in the range +5 to +7 pp m. A Ca2+ titration of N-15-labeled apo-calmodulin was performed using 2D H-1-N-15 HSQC NMR spectra. The changes in the N-15 chemical shifts and intensities for the peaks corresponding to the NH groups of resid ues in position 8 of the EF-hand loops allowed the amount of metal bou nd at sites II, III and IV to be monitored directly at partial degrees of saturation. The peak corresponding to site I could only be monitor ed at the beginning and end of the titration because of line broadenin g effects in the intermediate region of the titration. Sites III ana I v both titrate preferentially and the results demonstrate clearly that sites in either domain fill effectively in parallel, consistent with a significant positive intradomain cooperativity of calcium binding.