Linkage of Protein Folding and Assembly to DNA Recognition

University Of Mississippi, University MS

Investigators

Abstract

The Cro repressor helps to regulate the master switch that controls the development of bacteriophage lambda. The lambda system has served as a paradigm for the control of gene expression circuitry. This project aims to explore the extent to which the physical characteristics of Cro determine the behavior of this model biological control circuit. A dimer of 66-residue Cro subunits is required to form a specific complex on its operator DNA site. Previous work has shown that two factors can limit the rate of assembly of Cro dimers, (1) proline isomerization and (2) competing folded monomeric conformations. In unstructured polypeptide chains, peptide bonds preceding proline residues in the dimer interface can adopt non-native configurations that slow the assembly of dimers by more than 10,000-fold (from milliseconds to hundreds of seconds). At equilibrium, exchange of subunits between dimers is also very slow (hundreds of seconds) even under conditions where dimers and monomers are roughly equally populated. This project seeks to extend these observations in three ways. First, the proline residues in the dimer interface will be replaced to determine if it is possible to streamline the folding process. Second the kinetics of assembly of protein dimers, will be monitored in the presence of DNA. Third a new fluorescent tracer assay will be developed to characterize monomeric species that are critical intermediates in the DNA binding process but rare under conditions that are typically used for protein stability measurements. Broader impact: The University of Mississippi is committed to strengthening the diversity of the workforce in science and engineering. Several programs are in place to recruit, train and retain future scientists from historically underrepresented groups and to foster interactions and the flow of information between researchers at the University of Mississippi and students in K-12 education. Using these opportunities, this project will continue to foster the participation of underrepresented minority students in research. In addition, concepts and materials developed during the course of the previous award have been applied to undergraduate and graduate education through integration into laboratory and lecture courses. Undergraduate and graduate students enrolled in the biochemistry laboratory course will be direct participants in the mutagenesis, screening, preliminary protein expression and purification of Cro variants constructed by combinatorial mutagenesis.