Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins

Abstract

BackgroundDisulfide engineering is an important biotechnological tool that has advanced a wide range of research. The introduction of novel disulfide bonds into proteins has been used extensively to improve protein stability, modify functional characteristics, and to assist in the study of protein dynamics. Successful use of this technology is greatly enhanced by software that can predict pairs of residues that will likely form a disulfide bond if mutated to cysteines.ResultsWe had previously developed and distributed software for this purpose: Disulfide by Design (DbD). The original DbD program has been widely used; however, it has a number of limitations including a Windows platform dependency. Here, we introduce Disulfide by Design 2.0 (DbD2), a web-based, platform-independent application that significantly extends functionality, visualization, and analysis capabilities beyond the original program. Among the enhancements to the software is the ability to analyze the B-factor of protein regions involved in predicted disulfide bonds. Importantly, this feature facilitates the identification of potential disulfides that are not only likely to form but are also expected to provide improved thermal stability to the protein.ConclusionsDbD2 provides platform-independent access and significantly extends the original functionality of DbD. A web server hosting DbD2 is provided at http://cptweb.cpt.wayne.edu/DbD2/.

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

“…A PDB file (entry 5DN9) of the three-dimensional (3D) structure model of Cbo FDH was submitted to Disulfide by Design (http://cptweb.cpt.wayne.edu/DbD2/index.php) (15). Residue A10 was selected to pair with C23, and residue I239 was selected to pair with C262 according to the size of dihedral angles, χ 3 (16).…”

“…Previous reports (4, 30) and an accurate structural model of Cbo FDH (31, 32) provide more direct and readily available information related to enzyme properties for site-directed mutagenesis. Computational design methods provide more precise guidance for the engineering of disulfide bonds (15, 16) and make mutations more efficient. According to the results of SDS-PAGE (see Fig.…”

The Structure of a BamA-BamD Fusion Illuminates the Architecture of the β-Barrel Assembly Machine Core

“…Therefore, single cysteines were introduced in BamA and BamD such that, according to the crystal structure, they could form an inter-subunit disulfide bond. The ecBamA-BamD model was analyzed with the server “Disulfide by Design” (Craig and Dombkowski, 2013), which identified ecBamA G374 and ecBamD V192 as residues that, when mutated to cysteine, would have distances and orientations compatible with inter-subunit disulfide bond formation according to the crystal structure. These mutations were incorporated into the pZS21 plasmid described above encoding His-tagged BamA(G374C) and BamD(V192C) to test for disulfide bond formation in the full length proteins in vivo .…”

Crystal Structure of BamB Bound to a Periplasmic Domain Fragment of BamA, the Central Component of the β-Barrel Assembly Machine

“…We thus sought to introduce cysteines in BamA and BamB that would result in a disulfide bond if the proteins interact as defined in the crystal structure. Analysis of the BamA-BamB interface with the program Disulfide by Design (29) did not identify any pair of residues that would have a favorable geometry to form an interchain disulfide when mutated to cysteine. Nevertheless, manual inspection of the interface showed that the 4.7-Å distance between the ␤-carbons of BamA Val-183 and BamB Ala-129 may allow disulfide bond formation across the interface even though the geometry was not ideal.…”