Proteins that need to be structured in their native state must be stable both against the unfolded ensemble and against incorrectly folded (misfolded) conformations with low free energy. Positive design targets the first type of stability by strengthening native interactions. The second type of stability is achieved by destabilizing interactions that occur frequently in the misfolded ensemble, a strategy called negative design. Here, we investigate negative design adopting a statistical mechanical model of the misfolded ensemble, which improves the usual Gaussian approximation by taking into account the third moment of the energy distribution and contact correlations. Applying this model, we detect and quantify selection for negative design in most natural proteins, and we analytically design protein sequences that are stable both against unfolding and against misfolding. Proteins 2013; 81:1102–1112. © 2013 Wiley Periodicals, Inc.

BackgroundProtein alignments are an essential tool for many bioinformatics analyses. While sequence alignments are accurate for proteins of high sequence similarity, they become unreliable as they approach the so-called 'twilight zone' where sequence similarity gets indistinguishable from random. For such distant pairs, structure alignment is of much better quality. Nevertheless, sequence alignment is the only choice in the majority of cases where structural data is not available. This situation demands development of methods that extend the applicability of accurate sequence alignment to distantly related proteins.ResultsWe develop a sequence alignment method that combines the prediction of a structural profile based on the protein's sequence with the alignment of that profile using our recently published alignment tool SABERTOOTH. In particular, we predict the contact vector of protein structures using an artificial neural network based on position-specific scoring matrices generated by PSI-BLAST and align these predicted contact vectors. The resulting sequence alignments are assessed using two different tests: First, we assess the alignment quality by measuring the derived structural similarity for cases in which structures are available. In a second test, we quantify the ability of the significance score of the alignments to recognize structural and evolutionary relationships. As a benchmark we use a representative set of the SCOP (structural classification of proteins) database, with similarities ranging from closely related proteins at SCOP family level, to very distantly related proteins at SCOP fold level. Comparing these results with some prominent sequence alignment tools, we find that SABERTOOTH produces sequence alignments of better quality than those of Clustal W, T-Coffee, MUSCLE, and PSI-BLAST. HHpred, one of the most sophisticated and computationally expensive tools available, outperforms our alignment algorithm at family and superfamily levels, while the use of SABERTOOTH is advantageous for alignments at fold level. Our alignment scheme will profit from future improvements of structural profiles prediction.ConclusionsWe present the automatic sequence alignment tool SABERTOOTH that computes pairwise sequence alignments of very high quality. SABERTOOTH is especially advantageous when applied to alignments of remotely related proteins. The source code is available at http://www.fkp.tu-darmstadt.de/sabertooth_project/, free for academic users upon request.