CONTROL OF PROTEIN NUCLEATION AND CRYSTALLITE GROWTH

拟议研究的目标是获得定量的 对小分子蛋白质成核和成核后生长的认识 蛋白质晶体(微晶)，以：(A)改善结晶 协议产生与结构完美匹配的晶体 用于蛋白质研究的最新X射线衍射技术 结构/功能研究和合理药物设计；(B)实现狭义 微晶尺寸分布，这对于稳定的持续是必不可少的 释放药物蛋白制剂，如胰岛素；和(C) 找到调节体内蛋白质结晶的方法，例如， 红细胞中血红蛋白C晶体的形成。具体的 利用四种球状蛋白模型所追求的目标是：1)获得 蛋白质晶体成核的基本机制 通过研究成核速率对蛋白质的依赖关系 浓度和溶液过饱和度。2)定义并量化 (I)可溶性添加剂，对每种蛋白质具有生物特异性，在 代表那些通常发生在 蛋白质溶液结晶；和(Ii)大小已知的颗粒 和专注力。3)基于在AIMS 1)和 2)，证明了蛋白质结晶成核可以控制在 在上述(A)-(C)方面取得改进。基本假设 这项工作的基础是从无机物中衍生出的成核概念 系统，可根据解决方案中的差异进行修改 相互作用和分子动力学，可以为蛋白质研究提供指导 成核作用。拟议中的调查的一个关键要素是一部小说 蛋白质成核的自动多细胞显微技术 学习。这项技术允许直接测量成核 速率和成核条件的定量关联。不像 以前的蛋白质成核研究，这些成核率 测定不是基于对分子的任何假设 结晶溶液中的相互作用。

The objective of the proposed research is to obtain quantitative understanding of protein nucleation and post-nucleation growth of small protein crystals (crystallites) in order to: (a) improve crystallization protocols yielding crystals of a structural perfection that matches state-of-the-art X-ray diffraction techniques used in protein structure/function studies and rational drug design; (b) achieve narrow crystallite size distributions, which are essential for steady sustained release of pharmaceutical protein preparations, such as insulin; and (c) find means to regulate protein crystallization in vivo, e.g., the formation of hemoglobin C crystals in red blood cells. The specific aims to be pursued using four model globular proteins are: 1) Obtain insight into the fundamental mechanisms of protein crystal nucleation through studies of the dependencies of the nucleation rate on protein concentration and solution supersaturation. 2) Define and quantify the effects of: (i) soluble additives, biospecific for each protein, in concentrations representative of those commonly occurring in crystallizing protein solutions; and (ii) particulates with known size and concentration. 3) Based on the insight obtained under Aims 1) and 2), demonstrate that protein crystal nucleation can be controlled to achieve improvements in the areas (a)-(c) above. The basic hypothesis underlying this work is that nucleation concepts derived for inorganic systems, subject to modifications for differences in solution interactions and molecular kinetics, can provide guidance in protein nucleation. A key element of the proposed investigation is a novel automated multi-cell microscopy technique for protein nucleation studies. This technique allows direct measurements of the nucleation rates and quantitative correlations to the nucleation conditions. Unlike previous protein nucleation studies, these nucleation rate determinations are not based on any assumptions about the molecular interactions in the crystallization solution.