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


Abstract

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. Study holds ProTherm entries: 3846, 3847, 3848, 3849 Extra Details: computer model building; DNA binding activity; cysteine;,stability

Submission Details

ID: 8AgrQwzr3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:23 p.m.

Version: 1

Publication Details
Sauer RT;Hehir K;Stearman RS;Weiss MA;Jeitler-Nilsson A;Suchanek EG;Pabo CO,Biochemistry (1986) An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor. PMID:3539184
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