FORCES GOVERNING PROTEIN RECOGNITION DYNAMICS

这项研究的目的是直接测量分子间作用力。 管理蛋白质识别的动态以及与其他蛋白质的联系 蛋白质、小分子和膜结合受体。为此，我们 将使用表面作用力装置直接测量分子 均匀取向的蛋白质单层与第二种蛋白质之间的作用力 或配位体单层。我们将重点关注三个主要的直接测量 影响蛋白质识别和功能的蛋白质结构方面： 即L)蛋白质的静电表面电势，2)长的- 引导分子间对接和分子间对接的分子间力 提高动力学缔合率，以及3)分子基础 糖基化或膜碳水化合物对蛋白质的扰动 膜结合受体大分子的识别。在第一个 在这项工作阶段，我们将直接测量静电表面 两种大鼠肝细胞色素b5的两个不同表面区的电位 变异体、cyt c的结合面和 两种抗荧光素独特型单抗的Fab‘片段。我们会 将我们的结果与理论上确定的静电势进行比较 与相同的蛋白质表面区相对应的场。在 第二阶段，我们将阐明所有构成非接触的相互 分子作用力，如静电和范德华力 测定蛋白质-配体结合率。我们还将建立 表面电荷极性在重定向反应失准中的意义 成对的。我们将确定活动站点和外部站点的影响 表面突变对分子间长距离识别力的影响。 在第三阶段，我们将把我们的研究扩展到第四纪以外 以考察蛋白质糖基化和蛋白质结构的影响 特异蛋白和非特异蛋白的膜糖脂 互动。我们将确定亲和素的分子效应 界面结合上的糖基化作用。我们还将确定影响 神经节苷脂对受体介导的膜间黏附的作用 神经节苷脂的特性(GM1和GT3)及其表面密度。 了解支配蛋白质识别的分子作用力是 对我们理解蛋白质的基本基础是必不可少的 功能。蛋白质作用力的定量测定 行为将促进对蛋白质功能的先验预测 以及具有增强OR的蛋白质的行为以及合理的工程 精心设计的活动。此外，这种测量将导致 对现有理论模型的提炼。

The aim of this research is to directly measure the inter molecular forces governing the dynamics of protein recognition and associations with other proteins, small molecules, and membrane bound receptors. To this end, we will use a surface forces apparatus to directly measure the molecular forces between uniformly oriented protein monolayers and a second protein or ligand monolayer. We will focus on direct measurements of three main aspects of protein structure that impact protein recognition and function: namely, the l) the protein electrostatic surface potentials, 2) the long- ranged inter molecular forces that guide inter molecular docking and enhance kinetic association rates, and 3) the molecular basis of glycosylation or membrane carbohydrate perturbations to protein recognition of macromolecules of membrane-bound receptors. In the first stage of this work, we will directly measure the electrostatic surface potentials of two distinct surface regions of two rat liver cyt b5 variants, the binding surface of cyt c, and the binding surfaces of the Fab' fragments of two monoclonal antifluorescyl IgG idiotypes. We will compare our results with theoretically determined electrostatic potential fields corresponding to the identical protein surface regions. in the second stage, we will elucidate all constituent non contact inter molecular forces such as electrostatic and van der Waals forces that determine protein-ligand association rates. We will also establish the significance of surface charge polarity in reorienting misaligned reactive pairs. We will determine the effects of both active site and external surface mutations on the long ranged inter molecular recognition forces. In the third stage, we will extend our studies beyond the quaternary protein structure to examine the effects of protein glycosylation and membrane glycolipids on both specific and nonspecific protein interactions. We will determine the molecular effects of avidin glycosylation on interfacial binding. We will also determine the impact of gangliosides on receptor-mediated inter membrane adhesion as a function of the ganglioside identity (GM1 and GT3) and of its surface density. Knowledge of the molecular forces governing protein recognition is essential to our understanding of the fundamental basis of protein function. Quantitative determinations of the forces dictating protein behavior will facilitate the a priori predictions of protein function and behavior as well as the rational engineering of proteins with enhanced or finely tailored activities. Furthermore, such measurements will lead to the refinement of existing theoretical models.