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

点击翻译按钮获取中文摘要

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. ***