A New Fluorescence Technique for Protein-Folding Studies

蛋白质折叠的新荧光技术研究未展开的蛋白质分子的异质集合应该表现出端到端距离的广泛的，接近高斯分布，与大量的简并构象一致，因此具有高的构型熵。相反，蛋白质在其天然状态下，其肽骨架具有明确的三维定向，在一定程度上，其侧链也具有明确的三维定向。折叠蛋白质的构象自由度远小于随机聚合物的构象自由度，因此，构型熵大大低于未折叠的整体。能量景观代表了一个异构的未折叠蛋白质集合转化为一个均匀折叠的集合，预计类似于一个漏斗。蛋白质折叠研究的一个持续的挑战是理解一个不均匀的未折叠多肽的集合如何演变成一个整齐折叠的蛋白质集合。开发合适的折叠探针是至关重要的。一个有效的折叠探针必须不仅报告未展开、中间和折叠形式的总体，而且还应该提供这些实体的结构同质性的信息。荧光蛋白折叠探针可以设计为多肽集合提供种群和异质性信息。偶极-偶极荧光能量转移（FET）从激发给体* D到受体A的速率随DA分离的逆六次方而变化。如果D和A被合并到一个蛋白质分子中，测量*D荧光衰减动力学可以用来提取DA距离的分布。在蛋白质折叠反应期间的荧光衰减动力学测量可以区分两态折叠机制和一个未折叠的集合不断演变成折叠的蛋白质。本研究计划的目标是开发用于评估蛋白质折叠从触发到形成的整个过程中的DA距离分布的仪器和方法。

ABSTRACT9876443Jay WinklerCalifomia Institute of TechnologyA New Fluorescence Technique for Protein-folding StudiesA heterogeneous ensemble of unfolded protein molecules should exhibit a broad, nearly Gaussian distribution of end-to-end distances, consistent with a large number of degenerate conformations and, hence, a high configurational entropy. Proteins in their native states, by contrast, have definite three-dimensional orientations of their peptide backbones and, to a certain extent, their side chains as well. The conformational freedom in folded proteins is far smaller than that of random polymers and, consequently, the configurational entropy is considerably lower than that of the unfolded ensemble. The energy landscape representing the conversion of a heterogeneous ensemble of unfolded proteins into a uniformly folded collection is expected to resemble a funnel. A continuing challenge in protein-folding research is understanding how a heterogeneous ensemble of unfolded polypeptides evolves into a collection of neatly folded proteins. The development of suitable probes of the folding process is of paramount importance. An effective folding probe must report not just on the populations of unfolded, intermediate, and folded forms, but also should provide information about the structural homogeneity of these entities.Fluorescent protein-folding probes can be engineered to provide both population and heterogeneity information for a polypeptide ensemble. The rate of dipole-dipole fluorescence energy transfer (FET) from an excited donor * D to an acceptor A varies as the inverse sixth power of the DA separation. If D and A are incorporated into a single protein molecule, measurements of *D fluorescence decay kinetics can be used to extract distributions of DA distances. Measurements of fluorescence decay kinetics during a protein-folding reaction can distinguish between a two-state folding mechanism and one in which the unfolded ensemble continuously evolves into the folded protein. The goal of this research program is the development of instrumentation and methods for evaluation of DA distance distributions during the entire course of protein folding from triggering to formation of folded protein.