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

Abstract

Background: Disulfide 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.

Results: We 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.

Conclusions: DbD2 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/.

Figure 1

Distribution of χ₃ torsion angles observed in 1505 native disulfide bonds found in 331 PDB protein structures. Peaks occur at -87 and +97 degrees.

Figure 2

Distribution of the disulfide bond energy calculated for 1505 native disulfide bonds in our survey set using the DbD2 energy function. The mean value is 1.0 kcal/mol, and the 90th percentile is 2.2 kcal/mol.

Figure 3

Correlation of the estimated and actual χ₃ torsion angles for the 1418 native disulfide bonds that were correctly identified with DbD2 and also were predicted to have the correct chirality. An R² value of 0.995 demonstrates accurate prediction of disulfide atomic coordinates.

Figure 4

Comparison of native residue B-factors in stabilizing and destabilizing engineered disulfide bonds. The native structures associated with engineered disulfides previously reported as stabilizing (S) or destabilizing (D), based on experimental evidence, were analyzed with DbD2. The mean B-factor for residues involved in stabilizing disulfide bonds was 31.6 compared with 16.5 for those involved in destabilizing bonds, P = 0.066.