Phosphorylation is a key regulator of protein and cellular function. In this review, we describe tools that enable access to homogeneously phosphorylated protein and discuss examples that demonstrate how they can be applied in functional studies.
To expand the scope of native chemical ligation (NCL) beyond reactions at cysteine, ligation auxiliaries are appended to the peptide N‐terminus. After the introduction of a pyridine‐containing auxiliary, which provided access to challenging junctions (proline or β‐branched amino acids), we herein probe the role of the pyridine‐ring nitrogen. We observed side reactions leading to preliminary auxiliary loss. We describe a new easy to attach β‐mercapto‐β‐(4‐methoxy‐2‐pyridinyl)‐ethyl (MMPyE) auxiliary, which 1) has increased stability; 2) enables NCL at sterically encumbered junctions (e. g., Leu‐Val); and 3) allows removal under mildly basic (pH 8.5) conditions was introduced. The synthesis of a 120 aa long peptide containing eight MUC5AC tandem repeats via ligation of two 60mers demonstrates the usefulness. Making use of hitherto unexplored NCL to tyrosine, the MMPyE auxiliary provided access to a head‐to‐tail‐cyclized 21‐mer peptide and a His6‐tagged hexaphosphorylated peptide comprising 6 heptapeptide repeats of the RNA polymerase II C‐terminal domain.
Post‐translational modifications affect protein biology under physiological and pathological conditions. Efficient methods for the preparation of peptides and proteins carrying defined, homogeneous modifications are fundamental tools for investigating these functions. In the case of mucin 1 (MUC1), an altered glycosylation pattern is observed in carcinogenesis. To better understand the role of MUC1 glycosylation in the interactions and adhesion of cancer cells, we prepared a panel of homogeneously O‐glycosylated MUC1 peptides by using a quantitative chemoenzymatic approach. Cell‐adhesion experiments with MCF‐7 cancer cells on surfaces carrying up to six differently glycosylated MUC1 peptides demonstrated that different glycans have a significant impact on adhesion. This finding suggests a distinct role for MUC1 glycosylation patterns in cancer cell migration and/or invasion. To decipher the molecular mechanism for the observed adhesion, we investigated the conformation of the glycosylated MUC1 peptides by NMR spectroscopy. These experiments revealed only minor differences in peptide structure, therefore clearly relating the adhesion behaviour to the type and number of glycans linked to MUC1.
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