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.

Generation of a Potent Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Antagonist by Engineering a Stable Form of the Receptor-associated Protein (RAP) D3 Domain

“…Generation of Stable RAP Molecules-The introduction of engineered disulfide bonds into proteins has been widely used to stabilize them (21,22), and computational methods have been developed that analyze the three-dimensional structure of a protein to choose optimal sites for the creation of disulfide bonds. To engineer a RAP molecule with a stable D3 domain, we rationalized that introducing a disulfide bond between helices 2 and 3 of this domain would lead to its stabilization.…”

Protein engineering. The design, synthesis and characterization of factitious proteins

“…If the design of novel proteins is to have any meaning, a goal for protein engineering must be the development of objective computer-based methods for constructing and visualizing the new structure (see Fletterick & Zoller, 1986, for mini-reviews of the subject). A start has clearly been made in this direction, but confident predictions are still only possible in the simplest cases: those of conservative and isosteric substitutions in a protein whose structure has been determined at a resolution of 0.2 nm or better (Warshel & Sussman, 1986;Pabo & Suchanek, 1986). Moving away from the idealized situation to the deduction of structures by homologies increases the uncertainty surrounding the consequences of substitutions, but the problem may still be tractable in favourable cases (Chothia & Lesk, 1986;White, 1986).…”

“…Also, for certain topics or proteins which have already been covered in extenso in readily accessible journals their coverage in this review will be abbreviated. Fortunately there are now a number of excellent accounts available of the techniques for directed mutagenesis in vitro required for protein engineering (Itakura et al, 1984;Botstein & Shortle, 1985;Smith, 1985Smith, , 1986Carter, 1986) as well as recent summaries or reports of progress in the modification of an integral membrane protein (Khorana et al, 1987) and transport proteins (Kaback, 1987), approaches to the alteration of immunoglobulin structure and function Sharon et al, 1986;Jones et al, 1986;Shulman et al, 1986;Roberts & Rees, 1986), efforts to modify the properties of DNA-binding proteins (Wharton & Ptashne, 1985;Sauer et al, 1986), problems associated with the expression ofheterologous proteins in bacteria (Marston, 1986), and the use of genetics, including site-directed mutagenesis, to explore the mechanics of protein secretion (Beckwith & Ferro-Novick, 1986;Lenhardt et al, 1986). Although the latter topic is arguably of great importance to the technology of the production of secreted factitious proteins, it is a major subject in its own right and will not be covered here.…”

Engineering of an intersubunit disulphide bridge in glutathione reductase from Escherichia coli

“…Both problems can be studied by the techniques of site-directed mutagenesis, but attempts at engineering disulphide bridges into proteins have mainly been confined to intrachain bridges and have met with varying degrees of success [3][4][5]. The only reported example of an engineered disulphide bridge between subunits is for the N-terminal domain of the dimeric A-repressor [6].…”