Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level

• Published in: PloS one 2018-06, Vol.13 (6), p.e0198699
• Main Authors: Hobson, Jessica J, Gallegos, Austin S, Atha, 3rd, Benjamin W, Kelly, John P, Lein, Christina D, VanOrsdel, Cailtin E, Weldon, John E, Hemm, Matthew R
• Format: Article
• Language: English
• Subjects: Alanine; Amino Acid Sequence; Amino Acid Substitution - genetics; Amino acids; Analysis; Bacteria; Biochemistry; Biology and Life Sciences; Chromosomes, Bacterial - genetics; Cytochrome; Cytochrome bd; Cytochromes - genetics; Cytochromes - isolation & purification; Cytochromes - metabolism; Cytochromes - physiology; E coli; Electron Transport Chain Complex Proteins - genetics; Electron Transport Chain Complex Proteins - isolation & purification; Electron Transport Chain Complex Proteins - metabolism; Electron Transport Chain Complex Proteins - physiology; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli - physiology; Escherichia coli Proteins - genetics; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Escherichia coli Proteins - physiology; Functional plasticity; Gene expression; Gene mutation; Genetic aspects; Genomes; Genomics; Hydrophobicity; Hydrophobins; Immunoblotting; Investigations; Medical research; Mutants; Mutation; Mutation - genetics; N-Terminus; Oxidoreductases - genetics; Oxidoreductases - isolation & purification; Oxidoreductases - metabolism; Oxidoreductases - physiology; Phenotypes; Physical Sciences; Proteins; Research and Analysis Methods; Scanning mutagenesis
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0198699

Small proteins are a new and expanding area of research. Many characterized small proteins are composed of a single hydrophobic α-helix, and the functional requirements of their limited amino acid sequence are not well understood. One hydrophobic small protein, CydX, has been shown to be a component of the cytochrome bd oxidase complex in Escherichia coli, and is required for enzyme function. To investigate small protein sequence specificity, an alanine scanning mutagenesis on the small protein CydX was conducted using mutant alleles expressed from the E. coli chromosome at the wild-type locus. The resulting mutant strains were assayed for CydX function. No single amino acid was required to maintain wild-type resistance to β-mercaptoethanol. However, substitutions of 10-amino acid blocks indicated that the N-terminus of the protein was required for wild-type CydX activity. A series of double mutants showed that multiple mutations at the N-terminus led to β-mercaptoethanol sensitivity in vivo. Triple mutants showed both in vivo and in vitro phenotypes. Together, these data provide evidence suggesting a high level of functional plasticity in CydX, in which multiple amino acids may work cooperatively to facilitate CydX function.