Peng S, Liu L P, Emili A Q, Deber C M
Division of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
FEBS Lett. 1998 Jul 10;431(1):29-33. doi: 10.1016/s0014-5793(98)00715-7.
The gene responsible for cystic fibrosis encodes a membrane protein--the 1480-residue cystic fibrosis transmembrane conductance regulator (CFTR)--in which membrane-based CF-phenotypic mutants alter pore structure and/or impair ion transport. We report the preparation in milligram quantities and conformational characterization of a polypeptide comprised of CFTR transmembrane (TM) segments 3-4, a putative 'helical hairpin' portion of the CFTR TM1-6 domain. The TM segment 3-4 of CFTR was expressed in E. coli as a fusion protein linked to the C-terminus of His-tagged thioredoxin. Nickel chelate affinity chromatography, followed by release from the carrier by digestion with thrombin protease, gave free CFTR(TM3-4). Monitoring of the folding properties and conformational state(s) of the TM3-4 polypeptide using circular dichroism spectroscopy indicated a partial alpha-helical conformation in aqueous buffer, with up to 30% increase in alpha-helical content observed in membrane-mimetic environments.
导致囊性纤维化的基因编码一种膜蛋白——由1480个氨基酸残基组成的囊性纤维化跨膜传导调节因子（CFTR），其中基于膜的CF表型突变体会改变孔结构和/或损害离子转运。我们报告了由CFTR跨膜（TM）片段3 - 4组成的多肽的毫克级制备及其构象表征，CFTR跨膜片段3 - 4是CFTR TM1 - 6结构域中一个假定的“螺旋发夹”部分。CFTR的TM片段3 - 4在大肠杆菌中作为与His标签硫氧还蛋白C末端相连的融合蛋白表达。通过镍螯合亲和色谱法，然后用凝血酶蛋白酶从载体上释放，得到游离的CFTR(TM3 - 4)。使用圆二色光谱监测TM3 - 4多肽的折叠特性和构象状态，结果表明在水性缓冲液中呈部分α - 螺旋构象，在模拟膜环境中观察到α - 螺旋含量增加高达30%。
