Rop is the simplest and most regular member of a family of proteins characterized by a bundle of four antiparallel helices. Rop is dimeric, each monomer being formed by two helices connected by a sharp bend. In this work we have extensively mutagenized three residues that form the connection between the two α-helices to ask whether the bend region contains any important folding information. The characterization of a collection of random mutants indicated that this structure is rather insensitive to amino acid substitutions and that most amino acids are tolerated in these positions by the Rop native structure. In order to identify the rare amino acid sequences that would prevent Rop from folding and/or dimerizing, we exploited the observation that Rop can functionally substitute the dimerization domain of the λ repressor. In fact plasmids expressing a hybrid protein formed by the amino-terminal domain of the λ repressor covalently linked to Rop, confer immunity to λ infection on their hosts. We have shown that this property depends on the ability of the Rop moiety to fold and dimerize. The analysis of 380 Rop mutants containing random amino acid sequences at positions 30, 31 and 32 allowed us to identify three mutant Rop proteins that are defective in dimerization, probably as a consequence of their inability to fold. In these mutants the tripeptides VED, VPD and YPD substitute the wild-type DAD at positions 30, 31 and 32. Other combinations of amino acids are found resulting in levels of immunity that are lower than the wild-type but still sufficient to prevent single plaque formation. This result suggests that a smaller proportion of the corresponding Rop protein reaches a thermodynamic and proteolytically stable dimeric state.
|Number of pages||10|
|Journal||Journal of Molecular Biology|
|Publication status||Published - 1994|
- Four helix bundle
- Gene fusion
ASJC Scopus subject areas