Plasticity In Protein Sequence-Function Relationships

Nonconserved sequences present a challenge for elucidating the relationship between sequence and function in proteins. This relationship was investigated in the biotin repressor (BirA) system, which serves as both an essential metabolic enzyme and a transcription regulator in a large number of eubacteria and archaebacteria. In its repressor function, the vitamin biotin regulates repressor assembly on the bioO DNA sequence by allosterically activating BirA dimerization. Although several loop segments in the protein function in dimerization, the sequences of these segments are not conserved in the bifunctional biotin repressor protein family. In this work we employed site-saturation mutagenesis coupled to a genetic screen to investigate the sequence constraints on the function of six amino acid residues distributed throughout these loop regions. These six residues have previously been shown to be sensitive to amino acid replacement in in vitro dimerization measurements. The genetic screen, performed using a plate-based assay, yielded candidates with a range of activities in repression relative to the wild type system, with the breadth of the range dependent on the amino acid position analyzed. Overall, 55% of the candidates showed phenotypes similar to wild type, 21% showed stronger repression and 23% weaker. In quantitative in vivo measurements of the dependence of transcription levels on external biotin concentration, more than half of the isolates showed dependencies less than or similar to that measured for the wild type system. Notably, the dynamic range in some of the in vivo repression curves was highly variable. Thus, for some variants the plate assay reports on both the dynamic range and the biotin concentration-dependence of repression. Overall, the data reveal that many sequences provide similar transcription regulatory activity, which may have been key to evolving bifunctionality in the bacterial biotin repressors.