Stabilizing impact of N-glycosylation on the WW domain depends strongly on the Asn-GlcNAc linkage.
N-glycans play important roles in many cellular processes and can increase protein conformational stability in specific structural contexts. Glycosylation (with a single GlcNAc) of the reverse turn sequence Phe-Yyy-Asn-Xxx-Thr at Asn stabilizes the Pin 1 WW domain by -0.85 ± 0.12 kcal mol(-1). Alternative methods exist for attaching carbohydrates to proteins; some occur naturally (e.g., the O-linkage), whereas others use chemoselective ligation reactions to mimic the natural N- or O-linkages. Here, we assess the energetic consequences of replacing the Asn linkage in the glycosylated WW domain with a Gln linkage, with two natural O-linkages, with two unnatural triazole linkages, and with an unnatural α-mercaptoacetamide linkage. Of these alternatives, only glycosylation of the triazole linkages stabilizes WW, and by a smaller amount than N-glycosylation, highlighting the need for caution when using triazole- or α-mercaptoacetamide-linked carbohydrates to mimic native N-glycans, especially where the impact of glycosylation on protein conformational stability is important.
Department of Chemistry and Biochemistry, Brigham Young University , Provo, Utah 84602, United States.
Brijesh K
BK
Pandey
Sebastian
S
Enck
Joshua L
JL
Price
D001216:Asparagine / Q000737:chemistry*; D002942:Circular Dichroism; D056445:Cytoplasmic Dyneins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
D011506:Proteins; D001216:Asparagine; C427758:DYNLL1 protein, human; D056445:Cytoplasmic Dyneins