Biophysical Characterization of Macromolecules

本项目旨在开发新的和改进现有的生物大分子表征方法，并将这些方法协同应用于大分子及其相互作用的研究。所采用的技术有分析性超离心、动态光散射、等温滴定量热法和表面等离子体共振生物传感。在分析型超离心中，通过直接边界分析和Lamm方程建模来模拟大分子的大小分布，在方法上取得了进一步的进展。事实证明，这导致了相对高分辨率的尺寸分布，这对蛋白质低聚物状态的分析非常有用。此外，我们继续发展了用沉降速度法对小分子和带电大分子进行水动力学表征的方法。本文综述了沉淀平衡超离心在分子相互作用研究中的应用。对于光学生物传感，我们进一步改进了一种方法，可以显着减少所需的样本量。这些方法被协同应用于几种蛋白质及其可逆相互作用的表征。一个主要的焦点是病毒蛋白的寡聚状态，包括HIV包膜蛋白gp120和gp140，单纯疱疹病毒主要衣壳蛋白VP5的自结合特性，以及轮状病毒非结构蛋白NSP2的结构/功能关系。我们继续用表面等离子体共振生物传感技术研究g蛋白亚基。我们已经开始对其他几种蛋白质进行表征，包括轮状病毒非结构蛋白NSP5、SPE4、TAF、calretinin、微管蛋白、MHC分子、t细胞受体和超抗原SPE-C。许多这些系统的特征已经完成，一些合作出版物正在印刷、提交和准备中。

The intent of this project is to develop new and improve existing methodology for the characterization of biological macromolecules, and to apply these methods collaboratively to the study of macromolecules and their interactions. Techniques employed are analytical ultracentrifugation, dynamic light scattering, isothermal titration calorimetry, and surface plasmon resonance biosensing. In analytical ultracentrifugation, further methodological advances have been made in the modeling of macromolecular size-distributions by direct boundary analysis and Lamm equation modeling. This proved to result in relative high-resolution size-distributions, which were highly useful for the analysis of protein oligomeric states. Also, we continued the development of methods for the hydrodynamic characterization of small molecules and charged macromolecules by sedimentation velocity methods. We have written a review on sedimentation equilibrium ultracentrifugation for the study of molecular interactions. For optical biosensing, we have further improved a method that allows a significant reduction of the required sample volume. These methods were collaboratively applied to the characterization of several proteins and their reversible interactions. One major focus were the oligomeric state of viral proteins, including the HIV envelope proteins gp120 and gp140, the self-association properties herpes simplex major capsid protein VP5, and the structure/function relationship of the nonstructural protein NSP2 of rotavirus. We have continued our study of G-protein subunits by surface plasmon resonance biosensing. We have started the characterization of several other proteins, including the rotavirus nonstructural protein NSP5, SPE4, TAF, calretinin, tubulin, MHC molecules, T-cell receptors, and the superantigen SPE-C. The characterization of many of these systems has been completed, and several collaborative publications are in press, submitted, and in preparation.