Tion whereas in the competing protein-protein interaction model the BirA-biotin domain interaction is long lived. An approach that distinguishes these models utilized the small peptides that are substrates for biotinylation by BirA (136). These peptides, which were isolated by screening large peptide libraries, are quite diverse in sequence and have as few as two residues (one being the reactive lysine residue) that are found in naturally biotinylated proteins (136). Due to their small sizes (14 residues is sufficient, (136, 137)) and diverse sequences, it seems unlikely that stable peptide-BirA complexes are made. If these sequences (attached to a partner protein) are expressed in E. coli they should derepress bio operon expression, the Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone web Pepstatin site bio-AMP competition model is supported. If they fail to derepress, but are efficiently biotinylated, then the competing protein-protein interaction model would be supported. Although the most studied of these peptides (Pep-85) is reported to be as good a biotin acceptor as the AccB biotin domain, this peptide remains enigmatic because it seems to lack structure in solution (137) and can only be biotinylated by BirA (124). Biotin ligases from six other organisms fail to use this peptide as a biotin acceptor, although these ligases readily utilize the AccB domain as a substrate (124, 138). Thus, if the most studied peptide sequence somehow mimicked structural attributes of AccB, the sequence should be biotinylated by BPLs other than BirA. However, BirA is the only ligase known to biotinylate the Pep-85 sequence. Two fusion proteins containing synthetic biotin accepting peptide sequences (one being Pep-85) were as efficient in derepression of bio operon transcription as the when the naturalEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPageacceptor, AccB-87, was the fusion partner (138). These results argue strongly against the competing protein-protein interaction model. As noted above the strict specificity of Pep-85 for BirA argues against the peptides being structural mimics of the natural acceptor domain. Moreover, even if this were somehow the case the the peptides would have to interacted with BirA:bio-AMP with the same binding strength and kinetics as that of the natural acceptor protein despite their small size and markedly diverged sequences. Indeed, the peptides lack several residues postulated to play important roles in forming the putative heterodimer and have other residues, some of which cannot participate in hydrogen bonding) in place of resides thought to play roles in heterodimer formation. It it follows that the rules governing biotinylation are markedly different for AccB and the peptide sequences (138). The possibility that the competing protein-protein interactions model is the regulatory switch seems extremely remote. The classical work on BirA mutants did not include BirA superrepressing mutants. These would be mutants that would repress transcription under all conditions including biotin limitation and apo-domain overexpression. Some of the possible classes of mutants are: (i) BirA proteins unable to bind the biotin acceptor protein, (ii) BirA proteins that bind the acceptor protein but are unable to biotinylate it, (iii) BirA proteins that form very tight homodimers (perhaps even in the absence of bio-AMP) and (iv) BirA proteins that cannot dissociate from the operator DNA. Some of the mutants might be genetically dominant. Most of these mutants could be no.Tion whereas in the competing protein-protein interaction model the BirA-biotin domain interaction is long lived. An approach that distinguishes these models utilized the small peptides that are substrates for biotinylation by BirA (136). These peptides, which were isolated by screening large peptide libraries, are quite diverse in sequence and have as few as two residues (one being the reactive lysine residue) that are found in naturally biotinylated proteins (136). Due to their small sizes (14 residues is sufficient, (136, 137)) and diverse sequences, it seems unlikely that stable peptide-BirA complexes are made. If these sequences (attached to a partner protein) are expressed in E. coli they should derepress bio operon expression, the bio-AMP competition model is supported. If they fail to derepress, but are efficiently biotinylated, then the competing protein-protein interaction model would be supported. Although the most studied of these peptides (Pep-85) is reported to be as good a biotin acceptor as the AccB biotin domain, this peptide remains enigmatic because it seems to lack structure in solution (137) and can only be biotinylated by BirA (124). Biotin ligases from six other organisms fail to use this peptide as a biotin acceptor, although these ligases readily utilize the AccB domain as a substrate (124, 138). Thus, if the most studied peptide sequence somehow mimicked structural attributes of AccB, the sequence should be biotinylated by BPLs other than BirA. However, BirA is the only ligase known to biotinylate the Pep-85 sequence. Two fusion proteins containing synthetic biotin accepting peptide sequences (one being Pep-85) were as efficient in derepression of bio operon transcription as the when the naturalEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPageacceptor, AccB-87, was the fusion partner (138). These results argue strongly against the competing protein-protein interaction model. As noted above the strict specificity of Pep-85 for BirA argues against the peptides being structural mimics of the natural acceptor domain. Moreover, even if this were somehow the case the the peptides would have to interacted with BirA:bio-AMP with the same binding strength and kinetics as that of the natural acceptor protein despite their small size and markedly diverged sequences. Indeed, the peptides lack several residues postulated to play important roles in forming the putative heterodimer and have other residues, some of which cannot participate in hydrogen bonding) in place of resides thought to play roles in heterodimer formation. It it follows that the rules governing biotinylation are markedly different for AccB and the peptide sequences (138). The possibility that the competing protein-protein interactions model is the regulatory switch seems extremely remote. The classical work on BirA mutants did not include BirA superrepressing mutants. These would be mutants that would repress transcription under all conditions including biotin limitation and apo-domain overexpression. Some of the possible classes of mutants are: (i) BirA proteins unable to bind the biotin acceptor protein, (ii) BirA proteins that bind the acceptor protein but are unable to biotinylate it, (iii) BirA proteins that form very tight homodimers (perhaps even in the absence of bio-AMP) and (iv) BirA proteins that cannot dissociate from the operator DNA. Some of the mutants might be genetically dominant. Most of these mutants could be no.
