The molecular determinants of protein structure and DNA recognition will be studied with engineered variants of the lambda Cro protein. The dimer interface of Cro, a beta ribbon consisting of a strand from each subunit, is crucial for folding, stability, and DNA recognition. Engineering and mutagenesis efforts will be focused in this region to identify and isolate the roles of particular residues and structural elements in each of the above functions. A family of a novel monomeric variants of Cro has been constructed in which the wild type dimer interface has been replaced with a designed beta-hairpin turn. Data from recent 2D NMR experiments are consistent with the presence of the beta hairpin. Crystals of a Cro monomer have been obtained which diffract to beyond 1.6 Angstroms. The structures, stabilities, and DNA binding activities of these proteins will be correlated with the sequences of amino acid residues introduced to form the turns. The structure and flexibility of elements of local structure will be quantified in terms of their roles in maintaining high effective concentrations of interacting groups in both a simple monomeric protein structure and a complex between a dimeric DNA binding protein and its symmetric DNA site. A combination of rational design, random mutagenesis and genetic selection will be used to build new proteins which meet specific functional criteria. These proteins will be characterized and used to address specific questions of protein structure and DNA recognition. How do different combinations of amino acid residues affect the stiffness and extension of a beta ribbon or the properties of a beta hairpin? What coupling free energy can be attributed to a particular linkage between identical subunits when bound to a dyad symmetric operator site? Can variant linkages be used to alter specificities not by changing the residues in direct contact with DNA but by modifying the framework from which they are suspended? Answers to questions like these will refine our understanding of protein structure and function.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
Application #
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Notre Dame
Schools of Arts and Sciences
Notre Dame
United States
Zip Code
Rupert, P B; Mollah, A K; Mossing, M C et al. (2000) The structural basis for enhanced stability and reduced DNA binding seen in engineered second-generation Cro monomers and dimers. J Mol Biol 296:1079-90
Jana, R; Hazbun, T R; Fields, J D et al. (1998) Single-chain lambda Cro repressors confirm high intrinsic dimer-DNA affinity. Biochemistry 37:6446-55
Mossing, M C (1998) Solution structure and dynamics of a designed monomeric variant of the lambda Cro repressor. Protein Sci 7:983-93
Jana, R; Hazbun, T R; Mollah, A K et al. (1997) A folded monomeric intermediate in the formation of lambda Cro dimer-DNA complexes. J Mol Biol 273:402-16
Albright, R A; Mossing, M C; Matthews, B W (1996) High-resolution structure of an engineered Cro monomer shows changes in conformation relative to the native dimer. Biochemistry 35:735-42
Mollah, A K; Aleman, M A; Albright, R A et al. (1996) Core packing defects in an engineered Cro monomer corrected by combinatorial mutagenesis. Biochemistry 35:743-8