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，通过直接边界分析对沉降速度数据进行建模的方法取得了实质性进展。这包括一种新的代数噪声分解方法，最小二乘g*(s)方法，以及对连续尺寸分布的分析。在分析区离心方面获得了额外的经验，这是一种沉降速度法，可以最大限度地减少所需的样品体积。同时，我们继续发展小分子的流体动力学表征方法。我们扩展了我们在动态光散射和等温滴定量热法方面的专业知识，并撰写了一篇关于光学生物传感应用的实验方法的综述。实验上，合作研究的一个主要焦点是膜受体和免疫学中重要的相关蛋白的相互作用，如t细胞受体、不同的NK受体、MHC分子、超抗原和fcrn受体。第二个重点是病毒蛋白，包括HIV-gp120和单纯疱疹病毒衣壳蛋白。进一步研究的系统是g蛋白亚基相互作用，钙调蛋白与神经粒蛋白的相互作用，以及核小体及其与HMG蛋白的相互作用。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。-生物物理学，结合，热力学，动力学

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, substantial methodological advances have been made in the modeling of sedimentation velocity data by direct boundary analysis. This includes a novel algebraic noise decomposition method, a least-squares g*(s) method, and the analysis of continuous size distributions. Additional experience was gained in analytical zone centrifugation, a sedimentation velocity method that minimizes the required sample volume. Also, we continued the development of methods for the hydrodynamic characterization of small molecules. We have expanded our expertise in dynamic light scattering and isothermal titration calorimetry, and have written a review on the experimental methods applied in optical biosensing. Experimentally, one major focus of the systems collaboratively studied has been the interactions of membrane receptors and related proteins important in immunology, such as the T-cell receptor, different NK receptors, MHC molecules, superantigen, and FcRn-receptor. A second emphasis was in viral proteins, including HIV-gp120 and herpes simplex capsid proteins. Further systems studied are G-protein subunit interactions, calmodulin interactions with neurogranin, as well as nucleosomes and their interaction with HMG proteins. The characterization of many of these systems has been completed, and several collaborative publications are in press, submitted, and in preparation. - biophysics, binding, thermodynamics, kinetics