MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and 
exhibits truncated O-glycosylation. Overexpression and altered glycosylation 
make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed 
against MUC1 frequently bind an immunodominant epitope that contains a single 
site for O-glycosylation. Glycosylation with tumor carbohydrate antigens such as 
the Tn-antigen (GalNAc-O-Ser/Thr) results in antibodies binding with higher 
affinity. One proposed model to explain the enhanced affinity of antibodies for 
the glycosylated antigen is that the addition of a carbohydrate alters the 
conformational properties, favoring a binding-competent state. The 
conformational effects associated with Tn glycosylation of the MUC1 antigen was 
investigated using solution-state NMR and molecular dynamics. NMR experiments 
revealed distinct substructures of the glycosylated MUC1 peptides compared with 
the unglycosylated peptide. Molecular dynamics simulations of the MUC1 
glycopeptide and peptide revealed distinguishing differences in their 
conformational preferences. Furthermore, the glycopeptide displayed a smaller 
conformational sampling compared with the peptide, suggesting that the 
glycopeptide sampled a narrower conformational space and is less dynamic. A 
comparison of the computed ensemble of conformations assuming random 
distribution, NMR models, and molecular dynamics simulations indicated that the 
MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly 
ensembles and that these ensembles were different from that of the random coil. 
Together, these data support the hypothesis that that conformational 
pre-selection could be an essential feature of these peptides that dictates the 
binding affinities to MUC1 specific antibodies.