Prêcheur B, Siffert O, Bârzu O, Craescu C T
Institut National de la Santé et de la Recherche Médicale U.91 & URA 607, Hôpital Henri Mondor, Créteil, France.
Eur J Biochem. 1991 Feb 26;196(1):67-72. doi: 10.1111/j.1432-1033.1991.tb15786.x.
The solution conformation of a synthetic peptide of 20 amino acids (P235-254) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase was studied by proton two-dimensional NMR spectroscopy and circular dichroism. Based on the standard techniques we have assigned all the resonances in the NMR spectrum to the corresponding protons of the peptide. Analysis of the secondary chemical shift distribution and of the nuclear Overhauser effect connectivities showed no evidence for a highly populated regular conformation but suggested the tendency to form an alpha-helix around the unique Trp residue. The propensity for a helical structure is in agreement with the results of circular dichroic spectroscopy showing a slight negative band at 222 nm which was cancelled by 6 M guanidine hydrochloride. Increasing amounts of 2,2,2-trifluoroethanol (up to 40%) increase considerably the helical population of the peptide as reflected in the circular dichroic spectra. Analysis of the present results shows that the free peptide P235-254 has the tendency to form a basic amphiphilic helix. The presence of two acid residues, Glu236 and Asp239, on the hydrophilic side of the alpha-helix, which is mainly composed by basic residues, may explain the lower affinity of this peptide for calmodulin as compared with other peptides derived from calmodulin-activated enzymes.
利用质子二维核磁共振波谱和圆二色性研究了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20个氨基酸的合成肽（P235 - 254）的溶液构象。基于标准技术，我们已将核磁共振谱中的所有共振峰归属于该肽相应的质子。对二级化学位移分布和核Overhauser效应连接性的分析表明，没有证据显示存在高度占优势的规则构象，但提示在唯一的色氨酸残基周围有形成α - 螺旋的倾向。螺旋结构的倾向与圆二色光谱结果一致，该光谱在222 nm处显示出轻微的负峰，此峰在6 M盐酸胍存在时消失。如圆二色光谱所示，增加2,2,2 - 三氟乙醇的量（高达40%）会显著增加该肽的螺旋构象比例。对目前结果的分析表明，游离肽P235 - 254有形成碱性两亲性螺旋的倾向。在主要由碱性残基组成的α - 螺旋亲水区存在两个酸性残基Glu236和Asp239，这可能解释了该肽与源自钙调蛋白激活酶的其他肽相比，对钙调蛋白的亲和力较低的原因。
