Lockwood Nathan A, Tu Raymond S, Zhang Zhiwen, Tirrell Matthew V, Thomas David D, Karim Christine B
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA.
Biopolymers. 2003 Jul;69(3):283-92. doi: 10.1002/bip.10365.
We have used synthetic lipidated peptides ("peptide-amphiphiles") to study the structure and function of isolated domains of integral transmembrane proteins. We used 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis to prepare full-length phospholamban (PLB(1-52)) and its cytoplasmic (PLB(1-25)K: phospholamban residues 1-25 plus a C-terminal lysine), and transmembrane (PLB(26-52)) domains, and a 38-residue model alpha-helical sequence as a control. We created peptide-amphiphiles by linking the C-terminus of either the isolated cytoplasmic domain or the model peptide to a membrane-anchoring, lipid-like hydrocarbon tail. Circular dichroism measurements showed that the model peptide-amphiphile, either in aqueous suspension or in lipid bilayers, had a higher degree of alpha-helical secondary structure than the unlipidated model peptide. We hypothesized that the peptide-amphiphile system would allow us to study the function and structure of the PLB(1-25)K cytoplasmic domain in a native-like configuration. We compared the function (inhibition of the Ca-ATPase in reconstituted membranes) and structure (via CD) of the PLB(1-25) amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB(1-25)K has no effect on Ca-ATPase (calcium pump) activity, even when tethered to the membrane in a manner mimicking its native configuration, and that the transmembrane domain of PLB is sufficient for inhibition of the Ca-ATPase.
我们使用合成的脂化肽（“肽两亲分子”）来研究完整跨膜蛋白分离结构域的结构和功能。我们采用9-芴甲氧羰基（Fmoc）固相肽合成法制备全长的受磷蛋白（PLB(1 - 52)）及其胞质结构域（PLB(1 - 25)K：受磷蛋白的1 - 25位残基加上一个C端赖氨酸）、跨膜结构域（PLB(26 - 52)），并制备了一个38个残基的α螺旋模型序列作为对照。我们通过将分离的胞质结构域或模型肽的C端连接到膜锚定的类脂烃尾上，创建了肽两亲分子。圆二色性测量表明，无论是在水悬浮液中还是在脂质双层中，模型肽两亲分子的α螺旋二级结构程度都高于未脂化的模型肽。我们推测肽两亲分子系统将使我们能够以类似天然的构象研究PLB(1 - 25)K胞质结构域的功能和结构。我们比较了PLB(1 - 25)两亲分子与PLB及其分离的跨膜和胞质结构域在功能（对重组膜中Ca - ATP酶的抑制作用）和结构（通过圆二色性）方面的差异。我们的结果表明，即使以模拟其天然构象的方式连接到膜上，胞质结构域PLB(1 - 25)K对Ca - ATP酶（钙泵）活性也没有影响，并且PLB的跨膜结构域足以抑制Ca - ATP酶。
