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All these peptides were modified at position 6 with constrained amino acids which are commercially available.
The approach relies on a HH substrate modified at position 1.1, proximal to the cleavage site, with 2-aminopurine (2AP), an intensely fluorescent adenosine isoster.
Based on the previous result, several naringenin derivatives modified at position 7 with bulky substituents were designed and synthesized, and their inhibitory effects on HCT116 human colon cancer cells were tested using a clonogenic assay.
Compound MG 2778 (8), a cyclopenta[a]phenantrene derivative, was identified as lead compound which was chemically modified at position 2 in order to obtain a small library for preliminary SARs.
Besides the main phosphorylation site at S10 we also find a smaller fraction of the monophosphorylated peptide to be modified at position S1 or S3 as indicated by the existence of an unmodified y12 ion (y12 Fig 5D) and a modified b6 ion ( b6*, Fig 5E).
Interestingly, the CII259-273 epitope could be posttranslationally modified (at position 264 and 270) by lysine hydroxylation and glycosylation.
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The identity of the acid/base catalyst of the Family 35 β-galactosidases from Xanthomonas manihotis (BgaX) has been confirmed as Glu184 by kinetic analysis of mutants modified at that position.
In contrast, Trichoderma does not modify C-3, but exclusively makes trichothecenes modified at backbone position C-4, including trichodermol.
Most prominently, Fusarium makes only products modified at backbone position C-3, such as deoxynivalenol and T-2 toxin.
We then normalized the counts at each position by the number of counts for all fragments that were ligated at the 3′ end of the RNA, giving the fraction modified at each position.
The second molecular contact within the glycosylated peptide that is not dispensable for TCR triggering is the side chain primary amino group of Hyl, because analogues chemically modified at that position were barely recognized by the T cells.
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