Perturbing the folding energy landscape of the bacterial immunity protein Im7 by site-specific N-linked glycosylation

• Main Authors: Chen, Mark (Contributor), Bartlett, Alice I. (Author), Nerenberg, Paul S. (Contributor), Friel, Claire T. (Author), Hackenberger, Christian P. R. (Contributor), Radford, Sheena E. (Author), Imperiali, Barbara (Contributor), Stultz, Collin M (Author)
• Other Authors: Harvard University- (Contributor), Massachusetts Institute of Technology. Department of Biology (Contributor), Massachusetts Institute of Technology. Department of Chemistry (Contributor), Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science (Contributor), Massachusetts Institute of Technology. Department of Physics (Contributor), Stultz, Collin M. (Contributor)
• Format: Article
• Language: English
• Published: National Academy of Sciences, 2011-07-14T18:35:22Z.
• Subjects: Article
• Online Access: Get fulltext

N-linked glycosylation modulates protein folding and stability through a variety of mechanisms. As such there is considerable interest in the development of general rules to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in the design and development of modified proteins with advantageous properties. In this study, expressed protein ligation is used to create site-specifically glycosylated variants of the bacterial immunity protein Im7 modified with the chitobiose disaccharide (GlcNAc-GlcNAc). Glycans were introduced at seven solvent exposed sites within the Im7 sequence and the kinetic and thermodynamic consequences of N-linked glycosylation analyzed. The ΔΔG° [delta delta G superscript 0 or degree symbol] values for glycan incorporation were found to range from +5.2 to -3.8 kJ·mol-1. In several cases, glycosylation influences folding by modulating the local conformational preferences of the glycosylated sequence. These locally mediated effects are most prominent in the center of α-helices where glycosylation negatively effects folding and in compact turn motifs between segments of ordered secondary structure where glycosylation promotes folding and enhances the overall stability of the native protein. The studies also provide insight into why glycosylation is commonly identified at the transition between different types of secondary structure and when glycosylation may be used to elaborate protein structure to protect disordered sequences from proteolysis or immune system recognition.
National Institutes of Health (U.S.) (GM039334)
National Science Foundation (U.S.) (0821391)
Biotechnology and Biological Sciences Research Council (Great Britain) (Grant BB/526502/1)
Biotechnology and Biological Sciences Research Council (Great Britain) (Grant 24/B17145)