An Engineered Intersubunit Disulfide Enhances the Stability and DNA Binding of the N-Terminal Domain of λ Repressor

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

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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
DOIs
StatePublished - Oct 1986

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ASJC Scopus subject areas

  • Biochemistry

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Sauer, R. T., Hehir, K., Stearman, R. S., 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. https://doi.org/10.1021/bi00368a024