Root Kyle T, Glover Kerney Jebrell
Department of Chemistry, Lehigh University, 6 E. Packer Ave., Bethlehem, PA 18015, USA.
Department of Chemistry, Lehigh University, 6 E. Packer Ave., Bethlehem, PA 18015, USA.
Biochim Biophys Acta. 2016 Apr;1858(4):682-8. doi: 10.1016/j.bbamem.2016.01.007. Epub 2016 Jan 14.
Caveolin-1 is a membrane protein that possesses an unusual topology where both N- and C-termini are cytoplasmic as a result of a membrane-embedded turn. In particular, proline 110 has been postulated to be the linchpin of this unusual motif. Using a caveolin-1 construct (residues 62-178) reconstituted into dodecylphosphocholine micelles with and without a cholesterol mimic, the changes that occurred upon P110A mutation were probed. Using far UV circular dichroism spectroscopy it was shown that cholesterol attenuated the helicity of caveolin-1, and that mutation of P110 to alanine caused a significant increase in the α-helicity of the protein. Near UV circular dichroism spectroscopy showed significant changes in structure and/or environment upon mutation that again were modulated by the presence of cholesterol. Stern-Volmer quenching and λ(max) analysis of tryptophan residues showed that the proline mutation caused W85 to become more exposed, W98 and W115 to become less exposed, and W128 showed no change. This finding provided evidence that regions proximal and far away from the proline are buried differentially upon its mutation and therefore this residue is strongly tied to maintaining the hydrophobic coverage along the caveolin-1 sequence. In the presence of cholesterol, the accessibilities of the two tryptophan residues that proceeded position 110 were altered much more significantly upon P110A mutation than the two tryptophans aft P110. Overall, this work provides strong evidence that proline 110 is critical for maintaining both the structure and hydrophobic coverage of caveolin-1 and that cholesterol also plays a significant role in modulating these parameters.
小窝蛋白-1是一种膜蛋白，其拓扑结构不同寻常，由于存在一个膜嵌入转角，其N端和C端均位于细胞质中。特别是，脯氨酸110被认为是这种不同寻常基序的关键。使用一种在有无胆固醇模拟物的情况下重构到十二烷基磷酸胆碱胶束中的小窝蛋白-1构建体（第62 - 178位氨基酸残基），探究了P110A突变后发生的变化。通过远紫外圆二色光谱表明，胆固醇减弱了小窝蛋白-1的螺旋度，并且脯氨酸110突变为丙氨酸导致该蛋白的α-螺旋度显著增加。近紫外圆二色光谱显示，突变后结构和/或环境发生了显著变化，而这些变化同样受到胆固醇存在的调节。色氨酸残基的斯特恩-沃尔默猝灭和λ(max)分析表明，脯氨酸突变导致W85变得更暴露，W98和W115变得不那么暴露，而W128没有变化。这一发现提供了证据，表明在脯氨酸突变时，靠近和远离脯氨酸的区域被不同程度地掩埋，因此该残基与维持小窝蛋白-1序列上的疏水覆盖密切相关。在存在胆固醇的情况下，P110A突变后位于110位之前的两个色氨酸残基的可及性变化比110位之后的两个色氨酸更显著。总体而言，这项工作提供了有力证据，表明脯氨酸110对于维持小窝蛋白-1的结构和疏水覆盖至关重要，并且胆固醇在调节这些参数方面也起着重要作用。
