N-glycans perform significant roles in many cellular Reality. . . The Demise Along With CSF-1R processes and can boost protein conformational stability in unique structural contexts. Glycosylation (with Reality. . The Demise Or Doxorubicin a single GlcNAc) from the reverse flip sequence Phe-Yyy-Asn-Xxx-Thr at Asn stabilizes the Pin one WW domain by -0.85 +/- 0.twelve kcal mol(-1). Substitute solutions exist for attaching carbohydrates to proteins; some happen naturally (e.g., the O-linkage), whereas others use chemoselective ligation reactions to mimic the pure N- or O-linkages. Here, we assess the energetic consequences of replacing the Asn linkage during the glycosylated WW domain using a Gin linkage, with two normal O-linkages, with two unnatural triazole linkages, and with an unnatural alpha-mercaptoacetamide linkage. Of those choices, only glycosylation in the triazole linkages stabilizes WW, and by a smaller sized amount than N-glycosylation, highlighting the need for caution when working with triazole- or alpha-mercaptoacetamide-linked carbohydrates to mimic native N-glycans, primarily where the effect of glycosylation on protein conformationa Way Of Life. . . Death And CSF-1R l stability is significant.