A New Fluorescence Technique for Protein-Folding Studies

杰伊·温克勒加州理工学院用于蛋白质折叠研究的一种新的荧光技术未折叠的蛋白质分子的异质系综应该表现出广泛的、接近高斯的末端到末端的距离分布，与大量简并的构象相一致，因此具有高的构象熵。相比之下，天然状态下的蛋白质，其多肽骨架和一定程度上的侧链都有明确的三维取向。折叠蛋白质的构象自由度远小于无规聚合物的构象自由度，因此，构象熵明显低于未折叠的系综。能量图景代表着一组不同种类的未折叠蛋白质转化为统一折叠的集合，预计将像一个漏斗一样。蛋白质折叠研究中的一个持续挑战是了解不同种类的未折叠多肽如何进化成一组整齐折叠的蛋白质。开发适合折叠过程的探头是至关重要的。一个有效的折叠探针不仅必须报告未折叠、中间和折叠形式的种群，而且还应该提供关于这些实体的结构同质性的信息。可以设计荧光蛋白质折叠探针来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)从激发的施主*D到受主A的速率与DA分离的反六次方有关。如果D和A被结合到单个蛋白质分子中，则可以使用*D荧光衰减动力学的测量来提取DA距离的分布。对蛋白质折叠反应过程中荧光衰减动力学的测量可以区分两种折叠机制，一种是未折叠的系综不断演化为折叠的蛋白质。这项研究的目标是开发一种仪器和方法，用于评估蛋白质折叠从触发到形成折叠蛋白质的整个过程中DA距离的分布。

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.