Munier H, Bouhss A, Gilles A M, Palibroda N, Bârzu O, Mispelter J, Craescu C T
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Arch Biochem Biophys. 1995 Jul 10;320(2):224-35. doi: 10.1016/0003-9861(95)90004-7.
This paper reports the solution conformation of a peptide (P196-267) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. P196-267 corresponding to the protein fragment situated between amino acid residues 196-267 was overproduced by a recombinant Escherichia coli strain. Its affinity for calmodulin is only one order of magnitude lower (Kd = 2.4 nM) than that of the whole bacterial enzyme (Kd = 0.2 nM). The proton resonances of the NMR spectra of P196-267 were assigned using homonuclear two-dimensional techniques (double-quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser enhancement spectroscopy) and a standard assignment procedure. Analysis of the nuclear Overhauser effect connectivities and the secondary shift distribution of C alpha protons along the sequence allowed us to identify the elements of regular secondary structure. The peptide is flexible in solution, being in equilibrium between random coil and helical structures. Two segments of 11 amino acids (situated between V215 and A225) and 15 amino acids (situated between L233 and A247) populate in a significant proportion the helix conformational state. The two helices can be considerably stabilized in a mixed solvent, trifluoroethanol/water (30/70), suggesting that the corresponding fragment in the intact protein assumes a similar secondary conformation. No elements of tertiary structure organization were detected by the present experiments. The conformational properties of the isolated calmodulin target fragment are discussed in relation with the available NMR and X-ray data on various peptides complexed to calmodulin.
本文报道了一种源自百日咳博德特氏菌腺苷酸环化酶钙调蛋白结合结构域的肽（P196 - 267）的溶液构象。对应于氨基酸残基196 - 267之间蛋白质片段的P196 - 267由重组大肠杆菌菌株过量表达。它对钙调蛋白的亲和力仅比整个细菌酶低一个数量级（Kd = 2.4 nM）（Kd = 0.2 nM）。使用同核二维技术（双量子滤波J相关光谱、全相关光谱和核Overhauser增强光谱）以及标准归属程序对P196 - 267的NMR光谱的质子共振进行了归属。对核Overhauser效应连接性以及沿序列的Cα质子的二级位移分布的分析使我们能够识别规则二级结构的元件。该肽在溶液中是灵活的，处于无规卷曲和螺旋结构之间的平衡状态。两段11个氨基酸（位于V215和A225之间）和15个氨基酸（位于L233和A247之间）在很大比例上处于螺旋构象状态。这两个螺旋在三氟乙醇/水（30/70）的混合溶剂中可以得到相当程度的稳定，这表明完整蛋白质中的相应片段呈现出类似的二级构象。本实验未检测到三级结构组织的元件。结合关于与钙调蛋白复合的各种肽的现有NMR和X射线数据，讨论了分离的钙调蛋白靶片段的构象性质。
