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

R. T. Sauer, K. Hehir, Robert Stearman, M. A. Weiss, A. Jeitler-Nilsson, E. G. Suchanek, C. O. Pabo

Research output: Contribution to journalArticle

131 Citations (Scopus)

Abstract

Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of λ 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.

Original languageEnglish (US)
Pages (from-to)5992-5998
Number of pages7
JournalBiochemistry
Volume25
Issue number20
StatePublished - 1986
Externally publishedYes

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Disulfides
Dimers
Denaturation
DNA
Cysteine
Mutagenesis
Biochemistry
Dithiothreitol
Site-Directed Mutagenesis
Computer Simulation
Urea
Proteins
Substitution reactions
Hot Temperature

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sauer, R. T., Hehir, K., Stearman, R., Weiss, M. A., Jeitler-Nilsson, A., Suchanek, E. G., & Pabo, C. O. (1986). An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of λ repressor. Biochemistry, 25(20), 5992-5998.

An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of λ repressor. / Sauer, R. T.; Hehir, K.; Stearman, Robert; Weiss, M. A.; Jeitler-Nilsson, A.; Suchanek, E. G.; Pabo, C. O.

In: Biochemistry, Vol. 25, No. 20, 1986, p. 5992-5998.

Research output: Contribution to journalArticle

Sauer, RT, Hehir, K, Stearman, R, Weiss, MA, Jeitler-Nilsson, A, Suchanek, EG & Pabo, CO 1986, 'An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of λ repressor', Biochemistry, vol. 25, no. 20, pp. 5992-5998.
Sauer, R. T. ; Hehir, K. ; Stearman, Robert ; Weiss, M. A. ; Jeitler-Nilsson, A. ; Suchanek, E. G. ; Pabo, C. O. / An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of λ repressor. In: Biochemistry. 1986 ; Vol. 25, No. 20. pp. 5992-5998.
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AU - Pabo, C. O.

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