Structurial studies of thelac repressor using homology modeling and combinatorial mutations
Nichols, Jeffry Curtis
Doctor of Philosophy
The core domain (residues 62-323) of the regulatory protein lac repressor has been aligned to several sugar binding proteins of known structure. The overall homology based on two separate matrix scoring systems (minimum base change per codon and amino acid homology per residue) is significant. Similarly, the predicted secondary structure of the repressor exhibits excellent agreement with the known secondary structures of the sugar binding proteins. Using this primary sequence alignment, the tertiary structure of the core domain of lac repressor was modeled using the structures of the sugar binding proteins as templates. Further refinements of this model using the purine repressor provided improvement in regions not well defined based on the sugar binding proteins. Important residues involved in operator and sugar binding and in protein assembly have been identified using genetic methods, and placement of these residues in the model is consistent with their known function. The recent solution of the crystallographic structure confirms the elements of this homology model. This approach provides an effective means to visualize the core domain of the lac repressor and to interpret the mutational data for specific residues. The availability of models of this type provides a structural basis for rational design of experiments. Mutations that affect both monomer-monomer and dimer-dimer subunit interfaces have been combined to generate a new family of mutant proteins designed to explore the role of subunit interactions in this regulatory protein. Combination of apolar substitutions at residue 84 and C-terminal deletions to generate dimer results in mutant repressor proteins with increased stability, even in the presence of Tyr282Asp, a mutation that generates monomer in the wild-type background. Operator binding studies demonstrate the linkage between dimer formation and operator binding and confirm that dimers are the minimal unit required for repression. The nature of the side chain at position 84 not only influences subunit association and stability, but also appears to contribute to subunit communication and DNA binding domain orientation. Placement of the sites of these mutations in the homology model of the lac repressor is consistent with the characteristics found for these proteins.