The goals of this project are to understand how a protein binds to single-stranded nucleic acids and to understand and exploit the additivity of effects often resulting from multiple amino acid changes in a protein on its structure and function. Single-stranded nucleic acid- binding proteins play roles in key cellular processes such as DNA replication, recombination, and control of RNA translation. If the ways in which proteins interact with single-stranded nucleic acids were understood in detail, then it might be possible to modify the functions of these classes of proteins in a target fashion. This would be important in the treatment of diseases due to deficiencies in the function of these proteins. Proteins are very complex macromolecules, and engineering their properties in a completely rational manner is exceptionally difficult because the effects of changing the amino acid sequence of a protein cannot be predicted in detail. If functional and structural effects of amino acid substitutions in a protein could be accurately predicted for certain classes of multiple mutations based on the effects of the constituent single amino acid substitutions, then this process could be simplified. Such a simplification could greatly reduce the time and experimentation required to obtain a protein with a desired set of characteristics, such as a certain stability and affinity for a particular sequence of nucleic acid. The first part of this project is directed towards understanding how gene V protein interacts with single- stranded nucleic acids and how the protein preferentially recognizes a specific sequence of RNA and the cognate DNA. This is to be accomplished by determining crystal structures of complexes formed between gene V protein and oligonucleotides that bind specifically and non-specifically to the protein. We have already obtained high-quality co-crystals of two gene V protein-oligonucleotide complexes that diffract X-rays to resolutions of 3.0 angstroms and 3.4 angstroms, respectively. The goal of the second part of the project is to broaden our understanding of the additivity of effects of mutations on the structure and properties of a protein. This understanding of additivity is to be obtained by examining the effects of single and double mutations in gene V protein on the structure and properties of the protein, focusing on the relationship between the extent of additivity of effects of two mutations and the regions structurally affected by the mutations when made individually. The third part of this project is aimed at developing techniques that will allow macromolecular crystallographic experiments to be analyzed more accurately. We expect that the results of this project will have a substantial impact in the field of biotechnology and in the treatment of human disease.
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