The most attractive and methodologically convenient way to enhance protein stability is via the introduction of disulphide bond(s). However, the effect of the artificially introduced SS-bond on protein stability is often quite unpredictable. This raises the question of how to choose the protein sites in an intelligent manner, so that the ‘fastening’ of these sites by the SS-bond(s) would provide maximal protein stability. We hypothesize that the successful design of a stabilizing SS-bond requires finding highly mobile protein regions. Using GFP as an illustrative example, we demonstrate that the knowledge of the peculiarities of the intramolecular hydrophobic interactions, combined with the understanding of the local intrinsic disorder propensities (that can be evaluated by various disorder predictors, e.g., PONDRFIT), is sufficient to find the candidate sites for the introduction of stabilizing SS-bridge(s). In fact, our analysis revealed that the insertion of the engineered SS-bridge between two highly flexible regions of GFP noticeably increased the conformational stability of this protein toward the thermal and chemical unfolding. Therefore, our study represents a novel approach for the rational design of stabilizing disulphide bridges in proteins.

Recently, we designed a short α‐helical fibril‐forming peptide (αFFP) that can form α‐helical nanofibrils at acid pH. The non‐physiological conditions of the fibril formation hamper biomedical application of αFFP. It was hypothesized that electrostatic repulsion between glutamic acid residues present at positions (g) of the αFFP coiled‐coil sequence prevent the fibrillogenesis at neutral pH, while their protonation below pH 5.5 triggers axial growth of the fibril. To test this hypothesis, we synthesized αFFPs where all glutamic acid residues were substituted by glutamines or serines. The electron microscopy study confirmed that the modified αFFPs form nanofibrils in a wider range of pH (2.5–11). Circular dichroism spectroscopy, sedimentation, diffusion and differential scanning calorimetry showed that the fibrils are α‐helical and have elongated and highly stable cooperative tertiary structures. This work leads to a better understanding of interactions that control the fibrillogenesis of the αFFPs and opens opportunities for their biomedical application.

The most complex problem in studying multi-state protein folding is the determination of the sequence of formation of protein intermediate states. A far more complex issue is to determine at what stages of protein folding its various parts (secondary structure elements) develop. The structure and properties of different intermediate states depend in particular on these parts. An experimental approach, named μ-analysis, which allows understanding the order of formation of structural elements upon folding of a multi-state protein was used in this study. In this approach the same elements of the protein secondary structure are “tested” by substitutions of single hydrophobic amino acids and by incorporation of cysteine bridges. Single substitutions of hydrophobic amino acids contribute to yielding information on the late stages of protein folding while incorporation of ss-bridges allows obtaining data on the initial stages of folding. As a result of such an μ-analysis, we have determined the order of formation of beta-hairpins upon folding of the green fluorescent protein.