Kahana E, Gratzer W B
Medical Research Council Muscle and Cell Motility Unit, King's College, London, U.K.
Biochemistry. 1995 Jun 27;34(25):8110-4. doi: 10.1021/bi00025a017.
Fragments of the rod domain of dystrophin, which consists of spectrin-like repeating sequences, have been prepared by expression in Escherichia coli. The phasing established earlier for the dystrophin rod, as well as for Drosophila spectrin and smooth muscle alpha-actinin, suggested a length of less than 113 residues for the dystrophin repeat that we have chosen. Fragments with a common N-terminus and lengths between 113 and 119 residues were prepared. The formation of the stable native tertiary fold could be recognized by resistance to proteolysis, the circular dichroism spectrum in the regions of both peptide and aromatic absorption bands and the resolution of the long-wavelength component in the tryptophan absorption spectrum. It was found that the critical length for folding was 117 residues: shortening the chain by 1 further residue resulted in loss of the capacity to form a defined tertiary structure. Residue 117 is a glutamine; replacement of this by a methionine residue did not impair the ability of the chain to enter the folded conformation, implying that it is the length of the C-terminal alpha-helix, rather than any specific side-chain interaction, that is critical in determining the stability of the native structure. The fragment of 119 residues forms a significantly more stable structure than that of 117. It appears that the minimum unit capable of forming the native fold extends some residues into the adjoining sequence repeat.
肌营养不良蛋白的杆状结构域由血影蛋白样重复序列组成，已通过在大肠杆菌中表达制备而成。先前为肌营养不良蛋白杆状结构域以及果蝇血影蛋白和平滑肌α-肌动蛋白确定的相位，表明我们选择的肌营养不良蛋白重复序列长度小于113个残基。制备了具有共同N端且长度在113至119个残基之间的片段。稳定天然三级折叠的形成可通过对蛋白水解的抗性、肽吸收带和芳香族吸收带区域的圆二色光谱以及色氨酸吸收光谱中长波长成分的分辨率来识别。发现折叠的关键长度为117个残基：链再缩短1个残基会导致形成确定三级结构的能力丧失。第117位残基是谷氨酰胺；用甲硫氨酸残基取代该残基不会损害链进入折叠构象的能力，这意味着决定天然结构稳定性的关键是C端α-螺旋的长度，而不是任何特定的侧链相互作用。119个残基的片段形成的结构比117个残基的片段稳定得多。似乎能够形成天然折叠的最小单元向相邻序列重复延伸了一些残基。
