Purification and Analysis of Single-Stranded Binding Protein Tail Mutants

Single stranded binding protein (SSB) is a prokaryotic DNA protein that binds to single stranded DNA during times when the DNA is rendered from its double stranded form during times of genetic recombination or DNA damage in order to stabilize and protect it from further unnecessary harm. The protein exists as a tetramer with each monomer being made of an N-terminal and Cterminal domain. The C-terminal domain is made of two smaller sub-domains, both of which have yet to resolve properly in a crystal structure, named the intrinsically disordered linker and the acidic tip, with limited understanding on how they function and relate to other proteins and SSB itself. Due to the disordered nature of its C-terminal domain limiting the ability to yield a concise crystal structure, much of the function and nearly all of the structure of the C-terminal domain has yet to be identified. While some function has been determined for these disordered regions, its relationship with other binding partners, DNA, and itself have yet to be fully determined. Through the use of SSB mutants D4A4( where aspartic acid residues 191, 193, 194, and 195 are changed to alanines) and ΔC8 (where residues 191 through 198 are removed) in purification experiments and analytical techniques such as size exclusion chromatography, x-ray crystallography, and multi-angle light scattering, some potential hypotheses were built based on the resulting data. Overall, D4A4 showed a higher ability to crystallize compared to is counterpart but was plagued with difficulty when trying to concentrate to high amounts. It is hypothesized that D4A4 was more susceptible to degradation and/or aggregation compared to ΔC8 due to many issues seen throughout the purification process such as D4A4’s band in each gel splitting in two and increased difficulty concentrating, but no definite statement could be made regarding this possibility. Conditions tested during x-ray crystallography provided useful information for use in future crystallization attempts; however, because a crystal structure failed to be uncovered, more progress must be made to establish the best conditions for crystallization. Initial tests show that HDX is a valid approach for comparing these mutants to the full-length protein to uncover structural differences. The speculative function of these areas of interest can only be confirmed through more crystallography studies and additional experiments with binding partners and DNA used in tandem with other SSB mutants. Altogether, the purifications and initial results in this thesis lay a solid foundation for future work assessing the role of the SSB C-terminal domain.