FUNDAMENTAL PROCESSES OF MACROMOLECULAR CRYSTALLIZATION

为了充分了解生物大分子的行为， 蛋白质和核酸，我们必须知道它们的三维排列 它们的组成原子。 这种完整的结构信息 来自X射线和中子衍射研究，这需要大，高 所研究的分子的质量晶体。 由于最近的 衍射方法的进步，结构测定的瓶颈 已经变成了目标分子的结晶。 虽然 已经开发了各种技术，一些重要的类别， 分子仍然很难结晶;例如， 免疫球蛋白和整合膜蛋白（例如通道， 受体和能量转导蛋白）。 然而， 对大分子的基本过程系统研究较少， 结晶，因此没有制定指导方针的坚实基础， 有效的晶体生长。 我们的目标是填补这一空白。 具体的研究目标有两个方面： 结晶的模型系统，鸡蛋白色溶菌酶，并延长 这项工作的结果对其他蛋白质。 调查将涵盖 晶体生长的三个时间阶段：成核、后成核 生长和停止生长。 I.成核 准弹性光散射技术，以前 应用于研究溶菌酶的成核作用，将扩展到其他蛋白质 以测试其对优化条件的一般有效性， 成核 第一个实验将集中在困难的 使免疫球蛋白结晶，以确定问题是否在于 成核步骤或在随后的生长中。 二.成核后生长 将继续研究增长机制， 四氢溶菌酶晶体。 第一个目标是检验假设， 两种不同的机制涉及不同的制度， 过饱和 第二个问题涉及各种晶体生长的影响， 可能污染结晶溶液的蛋白质杂质。 一 第三个目的是研究晶体质量，如通过 衍射研究中可达到的最大分辨率与 生长条件和可能的生长机制，从而建立 优化指南。 三.停止增长。 已经观察到蛋白质晶体可以 在它们大到可以使用之前就停止生长了。 第一 要求是在明确规定的条件下，确定何时停止， 或更一般地说生长速率的降低。 下一个任务是 调查减少或停止的原因，特别是 这是由于晶体生长时无序的积累， 表面的（例如，杂质），或可能的蛋白质变性， 阻止进一步附着的表面。

To fully understand the behavior of biological macromolecules such as proteins and nucleic acids, we must know the three-dimensional arrangements of their constituent atoms. Complete structural information of this sort comes from X-ray and neutron diffraction studies, which require large, high quality crystals of the molecule under study. As a result of recent advances in diffraction methods, the bottleneck in structure determination has become the crystallization of the molecule of interest. Although a variety of techniques have been developed, some important classes of molecules have remained very difficult to crystallize; for example, whole immunoglobulin proteins and integral membrane proteins (e.g. channels, receptors, and energy transduction proteins). However, there has been very little systemic research on the fundamental processes of macromolecular crystallization, and thus no firm basis for developing guidelines for effective crystal growth. The goal is to fill this gap. The specific research aims are two-fold: to continue studies on crystallization of the model system, hen egg white lysozyme, and to extend results of this work to other proteins. Investigations will cover the three temporal stages of crystal growth: nucleation, postnucleation growth, and cessation of growth. I. Nucleation. The technique of quasi-elastic light scattering, previously applied to study nucleation in lysozyme, will be extended to other proteins to test its general effectiveness for the optimization of conditions for nucleation. The first experiments will focus on the difficult to crystallize immunoglobulins to determine whether the problem lies in the nucleation step or in later growth. II. Postnucleation Growth. Work will continue on the growth mechanism of tetragonal lysozyme crystals. A first aim is to test the hypothesis that two different mechanisms are involved in different regimes of supersaturation. A second concerns the effect on crystal growth of various protein impurities which may contaminate crystallization solutions. A third aim is to investigate whether crystal quality, as measured by the maximum resolution attainable in diffraction studies, is correlated with growth conditions and perhaps growth mechanism, thus establishing optimization guidelines. III. Cessation of Growth. It has been observed that protein crystals may cease to grow before they are large enough to be useful. The first requirement is to establish, under well-defined conditions, when cessation, or more generally reduction of growth rate, may occur. The next task is to investigate the cause of the reduction or cessation, in particular whether it is due to an accumulation of disorder as the crystal grows, a poisoning of the surface (e.g. by an impurity), or possible protein denaturation at the surface which inhibits further attachment.