FUNDAMENTAL PROCESSES OF MACROMOLECULAR CRYSTALLIZATION

要充分了解生物大分子的行为，如 蛋白质和核酸，我们必须知道三维排列 它们的组成原子。这类完整的结构信息 来自X射线和中子衍射研究，这需要很大、很高 正在研究的分子的高质量晶体。由于最近 结构测定的瓶颈--衍射法的研究进展 已经成为感兴趣的分子的结晶。尽管一个 已经开发了各种技术，一些重要的类 分子仍然很难结晶；例如，完整的 免疫球蛋白和完整的膜蛋白(例如， 受体和能量转导蛋白)。然而，已经有非常多的 对大分子基本过程的系统研究很少 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。 具体的研究目的有两个：继续研究 结晶模型体系，蛋清溶菌酶，并延伸 这项工作的结果对其他蛋白质也有影响。调查范围将包括 晶体生长的三个时间阶段：成核、成核后 生长和停止生长。 一、成核。准弹性光散射技术，以前 应用于溶菌酶成核的研究，将推广到其他蛋白质 为了测试其对优化条件的普遍有效性 成核作用。第一批实验将重点放在难以 使免疫球蛋白结晶以确定问题是否存在于 成核步骤或在以后的生长中。 二、成核后生长。将继续开展工作，研究 四方溶菌酶晶体。第一个目标是检验这一假设 两种不同的机制涉及到不同的制度 过饱和。第二个问题是关于不同种类的晶体生长的影响。 可能污染结晶溶液的蛋白质杂质。一个 第三个目标是调查晶体质量，如通过 在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、停止生长。已经观察到蛋白质晶体可能 在它们大到可以使用之前停止增长。第一 要求是，在明确定义的条件下，当停止， 或者更广泛地说，可能会出现增长速度的降低。下一项任务是 调查减少或停止的原因，特别是 这是由于随着晶体的生长，无序的积累，一种中毒 表面(例如，由于杂质)，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.