Combining thermostable mutations increases the stability of lambda repressor.
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 Two of these mutations are in helix 3, where Gly-46 and Gly-48 have been replaced by alanines [Hecht, M. H., et al.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.
Stearman
R S
RS
Department of Biophysics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Frankel
A D
AD
Freire
E
E
Liu
B S
BS
Pabo
C O
CO
D000595:Amino Acid Sequence; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D004268:DNA-Binding Proteins; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D012097:Repressor Proteins / Q000235:genetics; D013816:Thermodynamics; D014157:Transcription Factors / Q000235:genetics; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins