title: Perturbing the folding energy landscape of the bacterial immunity protein Im7 by site-specific N-linked glycosylation.
abstract: 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° 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.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Chen
authors.forename: Alice I
authors.initials: AI
authors.lastname: Bartlett
authors.forename: Paul S
authors.initials: PS
authors.lastname: Nerenberg
authors.forename: Claire T
authors.initials: CT
authors.lastname: Friel
authors.forename: Christian P R
authors.initials: CP
authors.lastname: Hackenberger
authors.forename: Collin M
authors.initials: CM
authors.lastname: Stultz
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
authors.forename: Barbara
authors.initials: B
authors.lastname: Imperiali
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics
chemical_list: D002352:Carrier Proteins; D004187:Disaccharides; C509253:E colicin-binding immunity protein Im7, E coli; D029968:Escherichia coli Proteins; C032438:chitobiose