Combining thermostable mutations increases the stability of .lambda. repressor

• Published in: Biochemistry (Easton) Vol. 27; no. 19; pp. 7571 - 7574
• Main Authors: Stearman, Robert S, Frankel, Alan D, Freire, Ernesto, Liu, Beishan, Pabo, Carl O
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-09-1988
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Calorimetry, Differential Scanning; DNA-Binding Proteins; Drug Stability; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Hot Temperature; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis. Repair; Mutation; Protein Denaturation; Repressor Proteins - genetics; Thermodynamics; Transcription Factors - genetics; Viral Proteins; Viral Regulatory and Accessory Proteins; Proteins; Temperature; Purification; Escherichia coli; Site directed mutagenesis; N terminal-Sequence; Bacteria; Mutation; Escherichieae; Enterobacteriaceae
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00419a059

We have combined three mutations previously shown to stabilize lambda repressor against thermal denaturation. Two of these mutations are in helix 3, where Gly-46 and Gly-48 have been replaced by alanines [Hecht, M. H., et al. (1986) Proteins: Struct., Funct., Genet. 1, 43-46]. The other mutation, which replaces Tyr-88 with cysteine, allows the protein to form an intersubunit disulfide bond [Sauer, R. T., et al. (1986) Biochemistry 25, 5992-5998]. Calorimetric measurements show that the two alanine substitutions stabilize repressor by about 8 degrees C, that the disulfide bond stabilizes repressor by about 8 degrees C, and that the triple mutant is 16 degrees C more stable than wild-type repressor.