SS-Stabilizing Proteins Rationally: Intrinsic Disorder-Based Design of Stabilizing Disulphide Bridges in GFP

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., PONDR-FIT), 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.

Key words: Green fluorescent protein; Differential scanning microcalorimetry; SS-bridge; SS-bond; Disulphide; Protein stability; Protein intrinsic disorder.

This article can be cited as:
B.S. Melnik, T.V. Povarnitsyna, A.S. Glukhov, T.N. Melnik, V.N. Uversky. SS-Stabilizing Proteins Rationally: Intrinsic Disorder-Based Design of Stabilizing Disulphide Bridges in GFP J. Biomol Struct Dyn 29(4), 815-824 (2012).

Bogdan S. Melnik1*
Tatiana V. Povarnitsyna1
Anatoly S. Glukhov1
Tatyana N. Melnik1
Vladimir N. Uversky2,3*

1Institute of Protein Research, RAS, 142290 Pushchino, Moscow Region, Russia
2Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, Florida 33612, USA
3Institute for Biological Instrumentation, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia


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