Biekofsky R R, Martin S R, Browne J P, Bayley P M, Feeney J
Molecular Structure Division, Physical Biochemistry Division, National Institute for Medical Research, London, U.K.
Biochemistry. 1998 May 19;37(20):7617-29. doi: 10.1021/bi9800449.
Examination of the NMR 15N chemical shifts of a number of EF-hand proteins 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 15N 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 density of the nitrogen atom. This deshielding effect is effectively independent of the binding of metal to the other site of an EF-hand pair, allowing the 15N shifts to be used as probes for site-specific occupancy of metal binding sites. In addition, a Ca2+-induced change in side-chain Halpha-Calpha-Cbeta-Hbeta torsion angle for isoleucine or valine residues in position 8 can also contribute to the deshielding of the amide 15N nucleus. This conformational effect occurs only in sites I or III and takes place upon binding a Ca2+ ion to the other site of 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 ppm. A Ca2+ titration of 15N-labeled apo-calmodulin was performed using 2D 1H-15N HSQC NMR spectra. The changes in the 15N chemical shifts and intensities for the peaks corresponding to the NH groups of residues in position 8 of the EF-hand loops allowed the amount of metal bound at sites II, III and IV to be monitored directly at partial degrees of saturation. The peak corresponding to site I could only be monitored at the beginning and end of the titration because of line broadening effects in the intermediate region of the titration. Sites III and IV 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.
对多种EF手型蛋白的核磁共振15N化学位移进行检测表明，典型EF手型环第8位残基（或假EF手型环第10位残基）的酰胺氮的位移值能很好地指示该位点的金属占据情况。第8位残基的NH与第7位残基的羰基共价相连，这是典型EF手型环中唯一与金属离子配位的主链羰基。当金属与该羰基配位时，由于O(7)=C(7)-N(8)酰胺基团的极化以及氮原子电子密度的相应降低，第8位的15N核会出现明显的去屏蔽效应（+4至+8 ppm）。这种去屏蔽效应实际上与金属与EF手型对另一位点的结合无关，从而使得15N位移可被用作金属结合位点特异性占据情况的探针。此外，第8位异亮氨酸或缬氨酸残基的Ca2+诱导的侧链Hα-Cα-Cβ-Hβ扭转角变化也会导致酰胺15N核的去屏蔽。这种构象效应仅发生在位点I或III，并且在Ca2+离子与EF手型对的另一位点（位点II或IV）结合时出现，而不论第一个位点是否被占据。这种效应的大小在+5至+7 ppm范围内。使用二维1H-15N HSQC核磁共振谱对15N标记的脱钙钙调蛋白进行了Ca2+滴定。EF手型环第8位残基NH基团对应峰的15N化学位移和强度变化使得能在部分饱和程度下直接监测位点II、III和IV处结合的金属量。由于滴定中间区域的谱线展宽效应，仅能在滴定开始和结束时监测对应位点I的峰。位点III和IV均优先滴定，结果清楚地表明任一结构域中的位点有效且并行填充，这与钙结合存在显著的正结构域内协同性一致。
