FORCES GOVERNING PROTEIN RECOGNITION DYNAMICS

本研究的目的是直接测量分子间力 控制蛋白质识别的动力学以及与其他 蛋白质、小分子和膜结合受体。为此我们 将使用表面力仪器直接测量分子 均匀取向的蛋白质单层和第二种蛋白质之间的力 或配体单层。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）蛋白质静电表面电位，2）长- 引导分子间对接的排列的分子间力， 增强动力学缔合速率，和3）的分子基础 糖基化或膜碳水化合物对蛋白质的扰动 识别膜结合受体的大分子。上 在本工作的第一阶段，我们将直接测量静电表面 两种大鼠肝细胞色素b5两个不同表面区域的电位 变体、cyt c的结合表面和cyt c的结合表面的结合。 两种单克隆抗荧光IgG独特型的Fab'片段。我们将 将我们的结果与理论上确定的静电势进行比较 字段对应于相同的蛋白质表面区域。在 第二阶段，我们将阐明所有组成的非接触式互联网 分子力，例如静电力和货车范德华力， 测定蛋白质-配体结合率。我们亦会设立 表面电荷极性在取向失调反应中意义 对.我们将确定活性位点和外部 表面突变对长程分子间识别力的影响。 在第三阶段，我们将把研究扩展到第四纪以外 蛋白质结构，以检查蛋白质糖基化的影响， 膜糖脂对特异性和非特异性蛋白质 交互.我们将确定抗生物素蛋白的分子效应 糖基化对界面结合的影响。 我们还将确定 神经节苷脂对受体介导的膜间粘附的作用 神经节苷脂的特性（GM 1和GT 3）及其表面密度。 关于控制蛋白质识别的分子力的知识是 对我们理解蛋白质的基础至关重要 功能决定蛋白质的力的定量测定 行为将有助于蛋白质功能的先验预测， 行为以及合理的蛋白质工程与增强或 精心定制的活动。此外，这种测量将导致 完善现有的理论模型。

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.