Membrane Protein Stability

描述(由申请人提供)：膜蛋白在许多细胞过程中起着重要的作用。这项研究的总体目标是了解蛋白质折叠过程的物理基础和膜蛋白质结构的生物物理基础。目前，对于膜蛋白来说，这两种机制都还不是很清楚。从热力学上讲，我们的工作解决了疏水性在膜蛋白折叠中所起的关键作用。在之前的授予期间，我们开发了一种新的疏水性标度，该标度使用真实的双层和真实的折叠膜蛋白来测量从水到膜中心的侧链转移自由能。基于这一成就，我们现在提议测试这一规模的一般性：(1)通过使用不同的膜蛋白支架测量侧链转移自由能；(2)通过确定双分子层中埋入的程度如何调制水到双分子层的转移自由能；以及(3)通过设计我们的蛋白质支架进行测量，以作为pH的函数，以解决可电离基团突变的能量后果如何随着电荷状态的变化而变化。从动力学上讲，我们在前一次拨款期间发现，大肠杆菌的脂头 基团可以起到能量势的作用，将膜蛋白从错误的(内)膜中分离出来，朝着正确的(外膜)位置进行分类。在第四个目标中，我们建议通过确定含有大肠杆菌的脂头基团诱导的折叠的动力学寿命、构象和激活能来剖析这种分类的生物物理基础。

DESCRIPTION (provided by applicant): Membrane proteins play essential roles in many cellular processes. The overall goal of the proposed research is to understand the physical basis of the protein folding process and the biophysical basis of membrane protein structures. At present, neither of these is well understood for membrane proteins. Thermodynamically, our work addresses the critical role that hydrophobicity plays in membrane protein folds. In the previous granting period, we developed a novel hydrophobicity scale that measures side-chain transfer free energies from water to the membrane center using a real bilayer and a real, folded membrane protein. Based on this achievement, we now propose to test the generality of this scale (1) By measuring side-chain transfer free energies using distinct membrane protein scaffolds; (2) By determining how extent-of-burial in the bilayer modulates water to bilayer transfer free energies; and (3) By engineering of our protein scaffold for measurements as a function of pH to address how the energetic consequences of ionizable group mutations vary with charge state. Kinetically, we discovered in the previous grant period that E. coli lipid head groups may act as energetic potentials that sort membrane proteins away from the wrong (inner) membranes and towards the correct (outer) membrane locations. We propose in a fourth aim to dissect the biophysical basis for this sorting by determining the kinetic lifetimes and conformations and activation energies to folding induced by E. coli-containing lipid head groups.