Reactivity-based analysis of domain structures in native replication protein A.
Replication protein A (RPA) is an essential heterotrimeric ssDNA binding protein that participates in DNA repair, replication, and recombination. Though X-ray and NMR experiments have been used to determine three-dimensional structure models of the protein's domain fragments, a complete RPA structural model has not been reported. To test whether the fragment structures faithfully represent the same portions in the native solution-state protein, we have examined the structure of RPA under biologically relevant conditions. We have probed the location of multiple amino acids within the native RPA three-dimensional structure using reactivity of these amino acids toward proteolytic and chemical modification reagents. In turn, we evaluated different structural models by comparing the observed native RPA reactivities with anticipated reactivities based on candidate structural models. Our results show that our reactivity analysis approach is capable of critically assessing structure models and can be a basis for selecting the most relevant from among alternate models of a protein structure. Using this analytical approach, we verified the relevance of RPA fragment models to the native protein structure. Our results further indicate several important features of native RPA's structure in solution, such as flexibility at specific locations in RPA, particularly in the C-terminal region of RPA70. Our findings are consistent with reported DNA-free structural models and support the role of conformational change in the ssDNA binding mechanism of RPA.
Nuss
Jonathan E
JE
Department of Biochemistry and Molecular Biology and Biomedical Sciences Ph.D. Program, Wright State University, Dayton, Ohio 45435, USA.
Sweeney
Deacon J
DJ
Alter
Gerald M
GM
D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051716:Replication Protein A / Q000737:chemistry*; D013997:Time Factors
D005609:Free Radicals; D010455:Peptides; D051716:Replication Protein A; D004247:DNA