Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
J Phys Chem B. 2010 Feb 11;114(5):1925-31. doi: 10.1021/jp910185w.
Use of model transmembrane helices and lipid bilayers is a tractable and straightforward approach to obtaining thermodynamic information on fundamental processes of membrane protein folding. The insertion of transmembrane helices from an aqueous phase into membranes, the initial step in the folding process, is especially difficult to investigate because of the insolubility of helices in the aqueous phase. We report here the design of a soluble transmembrane helix, (KR)(5)-AALALAA-AALWLAA-AALALAA-C(NBD)-NH(2) (NBD, 7-nitrobenz-2-oxa-1,3-diazole), consisting of a transmembrane region (AALALAA)(3), a central guest residue (W), and an N-terminal charged tag (KR)(5). Circular dichroism and fluorescence spectroscopy revealed that the peptide dissolved in water as a monomer with the guest residue exposed to the solvent. After the addition of large unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, the peptide rapidly partitioned into the vesicles and assumed a transmembrane helix. The partitioning Gibbs free energy was estimated to be -34.2 kJ mol(-1) at 25 degrees C. The Trp-to-Gly substitution reduced the partitioning by approximately 1.6 kJ mol(-1). Thus, the transmembrane helix was found to be a useful template for thermodynamic measurements of the partitioning of amino acids from water to the hydrophobic core of membranes.
使用跨膜螺旋和脂质双层模型是获得膜蛋白折叠基本过程热力学信息的一种可行且直接的方法。由于螺旋在水相中的不溶性，跨膜螺旋从水相向膜中的插入（折叠过程的初始步骤）尤其难以研究。我们在此报告了一种可溶性跨膜螺旋 (KR)(5)-AALALAA-AALWLAA-AALALAA-C(NBD)-NH(2)（NBD，7-硝基苯并-2-氧杂-1,3-二唑）的设计，该螺旋由跨膜区 (AALALAA)(3)、中心客体残基 (W) 和 N 端带电标签 (KR)(5) 组成。圆二色性和荧光光谱表明，该肽在水中溶解为单体，客体残基暴露于溶剂中。在添加由 1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱组成的大单分子层囊泡后，肽迅速分配到囊泡中并形成跨膜螺旋。在 25°C 时，分配吉布斯自由能估计为-34.2 kJ mol(-1)。色氨酸到甘氨酸的取代使分配减少了约 1.6 kJ mol(-1)。因此，跨膜螺旋被发现是测量氨基酸从水到膜疏水区分配的热力学的有用模板。
