LCP-Tm: An Assay to Measure and Understand Stability of Membrane Proteins in a Membrane Environment
LCP-Tm: An Assay to Measure and Understand Stability of Membrane Proteins in a Membrane Environment
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DOI：
10.1016/j.bpj.2009.12.4296
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发表时间：
2010-04-21
影响因子：
3.4
通讯作者：
Cherezov, Vadim
中科院分区：
文献类型：
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作者：
Liu, Wei;Hanson, Michael A.;Cherezov, Vadim
膜蛋白的结构和功能研究受到其在天然膜环境外的稳定性差的限制。开发新的方法，以有效地稳定膜蛋白纯化后立即生物物理研究是重要的，可能是研究更具挑战性的人类目标的关键。脂质立方相（LCP）提供了一个合适的稳定基质研究膜蛋白的光谱和其他生物物理技术，包括获得高度有序的膜蛋白晶体的结构研究。我们已经开发了一种强大而准确的测定，LCP-T-m，用于测量嵌入在LCP基质中的膜蛋白的热稳定性。在其两种实施方式中，蛋白质变性之后要么是配体释放时内在蛋白质荧光的变化，要么是巯基结合报告染料的荧光增加，该染料测量埋藏在天然结构中的半胱氨酸的暴露。将LCP-T-m试验应用于工程改造的人β（2）-肾上腺素能受体和细菌视紫红质，揭示了许多增加LCP中蛋白质稳定性的因素。该测定具有指导蛋白质工程努力和鉴定稳定条件的潜力，所述稳定条件可以提高获得本质上不稳定的膜蛋白的高分辨率结构的机会。
Structural and functional studies of membrane proteins are limited by their poor stability outside the native membrane environment. The development of novel methods to efficiently stabilize membrane proteins immediately after purification is important for biophysical studies, and is likely to be critical for studying the more challenging human targets. Lipidic cubic phase (LCP) provides a suitable stabilizing matrix for studying membrane proteins by spectroscopic and other biophysical techniques, including obtaining highly ordered membrane protein crystals for structural studies. We have developed a robust and accurate assay, LCP-T-m, for measuring the thermal stability of membrane proteins embedded in an LCP matrix. In its two implementations, protein denaturation is followed either by a change in the intrinsic protein fluorescence on ligand release, or by an increase in the fluorescence of a thiol-binding reporter dye that measures exposure of cysteines buried in the native structure. Application of the LCP-T-m assay to an engineered human beta(2)-adrenergic receptor and bacteriorhodopsin revealed a number of factors that increased protein stability in LCP. This assay has the potential to guide protein engineering efforts and identify stabilizing conditions that may improve the chances of obtaining high-resolution structures of intrinsically unstable membrane proteins.