Protein Biophysics in Cells

积累的蛋白质化学知识大部分是在体外的稀溶液中收集的。下一个前沿领域是超越试管进入细胞。PI正在使用核磁共振波谱对活细菌进行真正的体内研究。本项目未来三年的细胞内核磁共振研究将涉及三种蛋白质：球状蛋白质、部分折叠蛋白质和内在无序蛋白质。球状蛋白以凝乳胰蛋白酶抑制剂2为代表。部分折叠的蛋白由细胞色素b5表示。内在无序的蛋白质将由-突触核蛋白表示。初步数据表明，所有这些体系都适用于细胞内核磁共振，包括酰胺1H交换和自旋松弛的研究。主要的假设是，大肠杆菌内部的环境影响着蛋白质的扩散、稳定性和内部动力学。有三个目标。(1)利用脉冲场梯度扩散实验确定细胞内的有效黏度和细胞内蛋白质的大小。(2)通过质子/氘交换实验确定细胞中蛋白质的稳定性。(3)通过监测自旋弛豫来确定细胞内蛋白质的内部动力学。更广泛的影响：该项目通过促进发现和理解，同时促进教学、培训和学习，将研究和教育结合起来。本项目将对研究生和本科生进行研究培训和指导。

The accumulated knowledge about protein chemistry has largely been collected in vitro in dilute solution. The next frontier involves moving beyond the test tube and into cells. The PI is performing truly in vivo studies in living bacteria by using NMR spectroscopy. In-cell NMR studies in the next three years of this project will involve three protein classes: globular proteins, partially folded proteins, and intrinsically disordered proteins. Globular proteins will be represented by chymotrypsin inhibitor 2. Partially folded proteins will be represented by apocytochrome b5. Intrinsically disordered proteins will be represented by alpha-synuclein. Preliminary data show that all these systems are amenable to in-cell NMR, including studies of amide 1H exchange and spin-relaxation. The main hypothesis is that the environment inside E. coli affects the diffusion, stability, and internal dynamics of proteins. There are three objectives. (1) Determine the effective viscosity inside cells and the size of proteins inside cells by using pulsed field gradient diffusion experiments.(2) Determine protein stability in cells by using proton/deuterium exchange experiments. (3) Determine the internal dynamics of proteins in cells by monitoring spin relaxation.Broader Impacts: This project integrates research and education by advancing discovery and understanding while promoting teaching, training, and learning. Both graduate students and undergraduates will receive research training and mentoring in this project.