Circular Dichroism of fd and Pf3 Single-Strand DNA-Binding Proteins

9405683 Gray这个项目的主要目标是阐明fd g5p的酪氨酸对CD光谱的贡献，fd g5p是一个由87个残基组成的单链DNA结合蛋白模型，其晶体结构最近被重新确定。这种蛋白质是a-折叠蛋白质，蛋白质的五种酪氨酸主宰了它的CD光谱。当蛋白质与多核苷酸结合时，酪氨酰229 nm CD带发生变化。已知有两种酪氨酸具有重要功能，一种参与DNA结合，另一种至少参与蛋白质与DNA结合时形成的协作性二聚体-二聚体接触。由于所有五种酪氨酸都倾向于在单链DNA结合蛋白中保守，它们都可能在功能上是重要的，尽管功能并不都是已知的。我们的目标是研究酪氨酸被保守取代的突变蛋白质。突变蛋白质光谱之间的差异，以及与DNA形成络合物时蛋白质光谱的变化，将被用来确定每个酪氨酸CD贡献的位置。研究结果应该为计算和理解酪氨酸对其他蛋白质圆二色谱的贡献提供一个基准。第二个目标是研究来自Pf3噬菌体的单链DNA结合蛋白，它属于丝状病毒的一个不同结构类别。Pf3是一种较小的蛋白质，有78个残基，其中只有3个是酪氨酸。对这种蛋白质的研究将有助于对丝状噬菌体单链DNA结合蛋白的光谱和结合特性有更全面的了解。最后，将利用小角X射线散射技术研究Fd和Pf3蛋白与单链DNA在溶液中形成的络合物。有些细菌病毒称为丝状病毒，成熟时首先形成细长的螺旋结构。这个中间螺旋结构包含了新制造的病毒dna基因组，它被包裹在一个由S单一蛋白质组成的蛋白质外壳中。这种特殊的病毒蛋白(DNA结合蛋白)来自一种病毒(FD病毒)，已经被许多技术研究得很好，被认为是同类蛋白中的一种模式蛋白。洛斯阿拉莫斯国家实验室的合作者T·特维利格博士提供了这种蛋白质的许多突变变体。突变的蛋白质和它们与DNA形成的结构与紫外光表现出有趣的相互作用，它们以某种方式偏振(圆偏振)。现在可以研究许多突变蛋白质与这种类型的偏振光的相互作用，这将使人们能够异常完整地解释哪些蛋白质成分具有光学现象。将对来自另一种病毒(Pf3病毒)的类似蛋白进行类似的研究。最后，蛋白质包裹的DNA基因组的螺旋结构将由它们散射X射线的方式来定义。后一项工作将与洛斯阿拉莫斯国家实验室的另一位合作者J.Trewhella博士一起完成。总之，对这些蛋白质和突变蛋白质的研究应该有助于解释其他DNA结合蛋白的性质，以及它们在丝状病毒复制过程中的作用。***

9405683 Gray The main goal of this project is to elucidate the tyrosyl contributions to the CD spectrum of the fd g5p, a model single- strand DNA-binding protein of 87 residues whose crystal structure has recently been redetermined. The protein is a -sheet protein, and the five tyrosines of the protein dominate its CD spectrum. The tyrosyl 229 nm CD band changes when the protein binds to polynucleotides. Two of the tyrosines are known to have important functions, one being involved in DNA-binding and at least one other being involved in cooperative dimer-dimer contacts that are formed as the protein binds to DNA. Since all five tyrosines tend to be conserved among the ssDNA-binding proteins, they all may be functionally important, although the functions are not all known. Our objective will be to study mutant proteins in which the tyrosines have been conservatively replaced. Differences between the spectra of the mutant proteins, and changes in the protein spectra upon forming complexes with DNA, will be used to determine the location of each tyrosine CD contribution. The results of the study should provide a benchmark for calculating and understanding the contributions of tyrosines to the CD spectra of other proteins. A second goal is to study the ssDNA-binding protein from Pf3 phage, which belongs to a different structural class of filamentous viruses. The Pf3 is a smaller protein of 78 residues, of which only three are tyrosines. A study of this protein should add to a more general understanding of the spectral and binding properties of the filamentous phage ssDNA-binding proteins. Finally, the complexes formed by the fd and Pf3 proteins with ssDNA will be investigated in solution by small-angle X-ray scattering techniques. %%% Some bacterial viruses, called filamentous viruses, mature by first forming a long, thin helical structure. This intermediate helical structure contains the newly-made viral DNA genome encased in a protein shell made of a s ingle type of protein. This special virus protein (DNA-binding protein) from one virus (fd virus) has been very well-studied by many techniques, and it is considered to be a model protein in its class. Many mutant variants of this protein are available from collaborator Dr. T. Terwilliger at Los Alamos National Laboratory. The mutant proteins and the structures they form with DNA exhibit interesting interactions with ultraviolet light beams they are polarized in a certain way (circularly polarized). The interactions of many mutant proteins with this type of polarized light can now be studied, which will allow an exceptionally complete interpretation of which protein components have the optical phenomenon. A similar study will be undertaken on the analogous protein from a different virus (Pf3 virus). Finally, the helical structures of the protein-encased DNA genomes will be defined by the manner in which they scatter X-rays. The latter work will be done with another collaborator at Los Alamos National Laboratory, Dr. J. Trewhella. In total, the study of these proteins and mutant proteins should help explain the properties of other DNA-binding proteins and how they act during the replication of the filamentous viruses. ***