Reconstitution and spectroscopic analysis of caveolin-1 residues 62-178 reveals that proline 110 governs its structure and solvent exposure.
Reconstitution and spectroscopic analysis of caveolin-1 residues 62-178 reveals that proline 110 governs its structure and solvent exposure.
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DOI：
10.1016/j.bbamem.2016.01.007
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发表时间：
2016-04
期刊：
影响因子：
--
通讯作者：
Glover KJ
中科院分区：
文献类型：
--
作者：
Root KT;Glover KJ
小窝蛋白-1是一种膜蛋白，其具有不寻常的拓扑结构，其中N-和C-末端都是细胞质，这是膜嵌入转向的结果。特别是脯氨酸110被认为是这个不寻常的基序的关键。使用小窝蛋白-1构建体（残基62-178）重建成十二烷基磷酸胆碱胶束与和没有胆固醇模拟物，发生P110 A突变的变化进行了探测。用远紫外圆二色性光谱表明，胆固醇减弱了小窝蛋白-1的螺旋度，而P110突变为丙氨酸引起蛋白质的α-螺旋度显著增加。近紫外圆二色光谱显示突变后结构和/或环境的显着变化，再次通过胆固醇的存在下调制。色氨酸残基的Stern-Volmer猝灭和λmax分析表明，脯氨酸突变导致W85暴露更多，W 98和W115暴露更少，W128没有变化。这一发现提供的证据表明，在其突变后，接近和远离脯氨酸的区域被不同地掩埋，因此该残基与保持沿着小窝蛋白-1序列的疏水覆盖沿着密切相关。在胆固醇存在下，P110 A突变后，110位前两个色氨酸残基的附加值比P110后两个色氨酸残基的附加值发生更显著的改变。总的来说，这项工作提供了强有力的证据表明，脯氨酸110是至关重要的结构和疏水覆盖的小窝蛋白-1，胆固醇也发挥了重要作用，在调节这些参数。
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 λmax 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.