Core packing defects in an engineered Cro monomer corrected by combinatorial mutagenesis

The crystal structure of an engineered monomer of the lambda Cro repressor shows unexpected expansion of the hydrophobic core of the protein and disorder of the five C-terminal residues [Albright et al. (1996) Biochemistry 35, 735-742]. This structural information has guided the construction of a second generation of monomeric Cro proteins by combinatorial mutagenesis of selected core and C-terminal residues. Clones were identified in a library of randomized cro genes by a genetic screen for protein accumulation in Escherichia coli. Sequencing of candidate genes followed by purification and analysis of their product proteins has identified alternative arrangements of hydrophobic core residues which result in substantial increases in thermal stability. In contrast, residue replacements at the C-terminus have minor effects on stability but may increase protein expression levels.