A fundamental challenge of protein engineering is the ability to consistently design proteins that will spontaneously fold into ‘native-like’ structures. Combinatorial de novo design offers a solution, by limiting the sequence space and helping in understanding sequence-stability relationships. One model system for combinatorial design is Rop, a 63-residue homodimer, whose regulatory function in <i>E. coli </i>is to control plasmid copy number. Rop consists of layers of heptad repeats (a-g), with hydrophobic residues occupying ‘a’ and ‘d’ positions, forming the hydrophobic core. Interestingly, a break in the pattern results in the surface exposure of an Arg residue at position ‘d’ and burial of a Phe residue at position ‘e’. We hypothesize that the inter-chain interaction between Arg and an Asp across the dimer interface is indispensable to the protein’s stability and topology and might explain the packing defect observed. We designed combinatorial libraries by randomizing Arg55 and Arg55/Phe56 with degenerate codons. The functional variants were selected by a cell-based screen, which couples Rop’s activity to GFP expression. Thermal and chemical melts from circular dichroism highlight the role of electrostatics between Arg and Asp. NMR spectroscopy suggests a possible switch in topology for some variants. Restoring heptad pattern at 55 and 56 positions might stabilize the protein by forming a conformation different from wild type. Further understanding of packing defects and topological differences will stem from X-ray crystallography and Gibbs-Helmholtz analysis. These results will further aid in our understanding of sequence-stability relationship in Rop and subsequently improve our knowledge of combinatorial protein design in general.

Understanding packing defects and loop interactions in the four-helix bundle protein, Rop

251st National Meeting (2016)

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