title: Structural correlates of the temperature sensitive phenotype derived from saturation mutagenesis studies of CcdB.
abstract: Temperature sensitive (ts) mutants are widely used to reversibly modulate protein function in vivo and to understand functions of essential genes. Despite this, little is known about the protein structural features and mechanisms responsible for generating a ts phenotype. Also, such mutants are often difficult to isolate, limiting their use. In this study, a library consisting of 75% of all possible single-site mutants of the 101-residue, homodimeric Escherichia coli toxin CcdB was constructed. Mutants were characterized in terms of their activity at two different temperatures and at six different expression levels. Of the total of 1430 single-site mutants that were screened, 231 (16%) mutants showed a ts phenotype. The bulk of these consisted of 120 ts mutants found at all 22 buried sites and 34 ts mutants at all seven active site residues involved in binding DNA gyrase. Of the remaining ts mutants, 16 were found at residues in van der Waals contact with active site residues, 36 were at partially buried residues, and 30 resulted from introduction of Pro. Thus virtually all ts mutants could be rationalized in terms of the structure of the native protein and without knowledge of folding pathways. Data were analyzed to obtain insights into molecular features responsible for the ts phenotype and to outline structure- and sequence-based criteria for designing ts mutants of any globular protein. The criteria were validated by successful prediction of ts mutants of three other unrelated proteins, TBP, T4 lysozyme, and Gal4.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Kanika
authors.initials: K
authors.lastname: Bajaj
authors.forename: Pooja C
authors.initials: PC
authors.lastname: Dewan
authors.forename: Purbani
authors.initials: P
authors.lastname: Chakrabarti
authors.forename: Devrishi
authors.initials: D
authors.lastname: Goswami
authors.forename: Bipasha
authors.initials: B
authors.lastname: Barua
authors.forename: Chetana
authors.initials: C
authors.lastname: Baliga
authors.forename: Raghavan
authors.initials: R
authors.lastname: Varadarajan
mesh_terms: D000595:Amino Acid Sequence; D001089:Arabinose / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D035181:TATA-Box Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D001089:Arabinose; D009113:Muramidase; D027081:DNA Gyrase