Zhang Yian-Biao, Howitt Jason, McCorkle Sean, Lawrence Paul, Springer Karen, Freimuth Paul
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Protein Expr Purif. 2004 Aug;36(2):207-16. doi: 10.1016/j.pep.2004.04.020.
Folding of the human coxsackie and adenovirus receptor immunoglobulin (Ig) variable-type domain (CAR D1) during overexpression in the Escherichia coli cytoplasm was shown previously to be partially rescued by fusion to a 22-residue C-terminal peptide. Here, peptide sequence features required for solubilization and folding of CAR D1 and similar Ig variable-type domains from two other human membrane proteins were investigated. Peptide extensions with net negative charge > -6 fully solubilized CAR D1, and approximately half of the peptide-solubilized protein was correctly folded. The Ig variable-type domains from human A33 antigen and myelin P-zero proteins were only partially solubilized by peptide extensions with net charge of -12, however, and only the solubilized P-zero domain appeared to fold correctly whereas the A33 domain formed soluble microaggregates of misfolded protein. Our results suggest a model where the large net charge of peptide extensions increases electrostatic repulsion between nascent polypeptides. The resulting decrease in aggregation rate can enable some polypeptides to fold spontaneously into their native protein conformations. Analysis of the solubility and folding status of sets of structurally homologous proteins, such as the Ig variable-type domains described here, during overexpression could provide insights into how amino acid and gene sequences influence the efficiency of spontaneous protein folding.
先前的研究表明，人柯萨奇病毒和腺病毒受体免疫球蛋白（Ig）可变型结构域（CAR D1）在大肠杆菌细胞质中过表达时，通过与一个22个残基的C末端肽融合，其折叠过程得到了部分挽救。在此，我们研究了CAR D1以及另外两种人类膜蛋白的类似Ig可变型结构域溶解和折叠所需的肽序列特征。净负电荷大于-6的肽延伸序列能使CAR D1完全溶解，并且大约一半的肽溶解蛋白能正确折叠。然而来自人A33抗原和髓鞘P-零蛋白的Ig可变型结构域仅被净电荷为-12的肽延伸序列部分溶解，并且只有溶解的P-零结构域似乎能正确折叠，而A33结构域形成了错误折叠蛋白的可溶性微聚集体。我们的结果提出了一个模型，其中肽延伸序列的大净电荷增加了新生多肽之间的静电排斥。由此导致的聚集速率降低可以使一些多肽自发折叠成其天然蛋白质构象。在过表达过程中分析一组结构同源蛋白（如此处所述的Ig可变型结构域）的溶解度和折叠状态，可以深入了解氨基酸和基因序列如何影响蛋白质自发折叠的效率。
