Li S C, Goto N K, Williams K A, Deber C M
Division of Biochemistry Research, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6676-81. doi: 10.1073/pnas.93.13.6676.
Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.
脯氨酸被确认为可溶性（球状）蛋白质中α-螺旋和β-折叠结构的强力破坏者。因此，在整合膜蛋白（尤其是转运蛋白）的假定跨膜螺旋中频繁出现脯氨酸残基，这带来了一个结构难题。我们认为，这种现象是由于给定氨基酸的结构倾向可能会因分子环境的变化而改变。为了在脯氨酸上验证这一假设，我们合成了通用序列为H2N-(Ser-LyS)2-Ala-Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH（Z = Ala和/或Pro）的模型肽。通过圆二色光谱在水性缓冲液、SDS、溶血磷脂酰甘油胶束和有机溶剂（甲醇、三氟乙醇和2-丙醇）中分析肽的构象。发现在脂质胶束和有机溶剂这两种模拟膜的环境中，脯氨酸的螺旋倾向大大增强。在2-丙醇中，脯氨酸在高温下相对于丙氨酸能稳定α-螺旋构象，这一观察结果与脯氨酸在水性介质中是最有效的α-螺旋破坏者这一学说相悖。在脱氧胆酸盐胶束中对单跨膜噬菌体IKe主要外壳蛋白的温度诱导构象转变进行的平行研究中，将含脯氨酸的野生型与Pro30→Ala突变体进行比较，发现脯氨酸能保护螺旋，但像在水中对模型肽那样有效地破坏β-折叠结构。脯氨酸破坏β-折叠的内在能力在孔蛋白的研究中进一步得到体现，在孔蛋白中发现脯氨酸被选择性地排除在跨膜β-折叠桶的核心之外。总体数据为预测和理解脯氨酸出现在膜环境中的结构后果提供了理论依据。
