This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The main goal of this project is to understand the evolutionary constraints and patterns that govern the natural selection of dynamic, flexible proteins by developing a more comprehensive picture of their rapidly interconverting structural ensembles. To accomplish this goal, we have selected a model system based on a conserved flexible linker from the 70 KDa subunit of replication protein A (RPA70). We are using this model system to determine if and how natural selection works to preserve the structure and function of dynamic, flexible proteins and protein domains. We have investigated the transient secondary structure and dynamics of the linkers from four RPA70 homologues and have observed the maintenance of a highly dynamic character throughout the course of evolution. Larger fragments of the human RPA70 homologue have revealed distinct interactions between the ends of the linker and the flanking folded domains. We are currently investigating the whether or not this is a general feature. We have also investigated the sequence conservation of more than 30 RPA70 homologues and hypothesize the linkers are evolving by insertion and deletion events as well as repeat expansion. Finally, Bayesian classifiers are being developed to identify dynamic, flexible protein structures based on chemical properties. While the presence and importance of dynamic flexible proteins and protein domains in nature is well established, this proposal marks the first systematic empirical investigation into their evolutionary constraints and patterns.