An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor.

Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of lambda repressor with cysteine. Computer model building had suggested that this substitution would allow formation of an intersubunit disulfide without disruption of the dimer structure [Pabo, C. O., & Suchanek, E. G. (1986) Biochemistry (preceding paper in this issue)]. We find that the Cys-88 protein forms a disulfide-bonded dimer that is very stable to reduction by dithiothreitol and has increased operator DNA binding activity. The covalent Cys88-Cys88' dimer is also considerably more stable than the wild-type protein to thermal denaturation or urea denaturation. As a control, Tyr-85 was replaced with cysteine. A Cys85-Cys85' disulfide cannot form without disrupting the wild-type structure, and we find that this disulfide bond reduces the DNA binding activity and stability of the N-terminal domain.

[1]  J. Berg,et al.  Systematic variation in DNA length yields highly ordered repressor-operator cocrystals. , 1985, Science.

[2]  R. Sauer,et al.  Phage lambda repressor revertants. Amino acid substitutions that restore activity to mutant proteins. , 1985, Journal of molecular biology.

[3]  Robert T. Sauer,et al.  Lambda repressor mutations that increase the affinity and specificity of operator binding , 1985, Cell.

[4]  T. Creighton,et al.  Kinetic role of a meta-stable native-like two-disulphide species in the folding transition of bovine pancreatic trypsin inhibitor. , 1984, Journal of molecular biology.

[5]  Alan R. Fersht,et al.  The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus) , 1984, Cell.

[6]  R. Sauer,et al.  Effect of single amino acid replacements on the thermal stability of the NH2-terminal domain of phage lambda repressor. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Brosius,et al.  Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. , 1983, Gene.

[8]  J. Vieira,et al.  A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. , 1982, Gene.

[9]  C. Pabo,et al.  The operator-binding domain of λ repressor: structure and DNA recognition , 1982, Nature.

[10]  M. Karplus,et al.  Fluctuations and averaging of proton chemical shifts in the bovine pancreatic trypsin inhibitor. , 1982, Biochemistry.

[11]  M Ptashne,et al.  Mechanism of action of the lexA gene product. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Mark Ptashne,et al.  Interactions between DNA-bound repressors govern regulation by the λ phage repressor , 1979 .

[13]  Mark Ptashne,et al.  Regulatory functions of the λ repressor reside in the amino-terminal domain , 1979, Nature.

[14]  R. Sauer,et al.  The lambda repressor contains two domains. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[15]  R. Sauer.  DNA sequence of the bacteriophage λ cI gene , 1978, Nature.

[16]  M. Ptashne,et al.  Maximizing gene expression on a plasmid using recombination in vitro , 1978, Cell.

[17]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[18]  W. Gilbert,et al.  Construction of plasmids carrying the cI gene of bacteriophage lambda. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[19]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[20]  F. Bovey,et al.  Calculation of Nuclear Magnetic Resonance Spectra of Aromatic Hydrocarbons , 1958 .

[21]  M. Smith,et al.  Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. , 1983, Methods in enzymology.