Site-selective labeling of endogenous proteins represents a major challenge in chemical biology, mainly due to the absence of unique reactive groups that can be addressed selectively. Recently, we have shown that surface-exposed lysine residues of two endogenous proteins and a peptide exhibit subtle changes in their individual reactivities. This feature allows the modification of a single residue in a highly site-selective fashion if kinetically controlled labeling conditions are applied. In order to broaden the scope of the "kinetically-controlled protein labeling" (KPL) approach and highlight additional applications, the water-soluble bioorthogonal reagent, biotin-TEO-azido-NHS (11), is developed which enables the site-selective introduction of an azido group onto endogenous proteins/peptides. This bioconjugation reagent features a biotin tag for affinity purification, an azido group for bioorthogonal labeling, a TEO (tetraethylene oxide) linker acting as a spacer and to impart water solubility and an N-hydroxysuccinimidyl (NHS) ester group for reacting with the exposed lysine residue. As a proof of concept, the native protein ribonuclease A (RNase A) bearing ten available lysine residues at the surface is furnished with a single azido group at Lys 1 in a highly site-selective fashion yielding azido-(K1)RNase A. The K1 site-selectivity is demonstrated by the combined application and interpretation of high resolution MALDI-ToF mass spectroscopy, tandem mass spectroscopy and extracted ion chromatography (XIC). Finally, the water soluble azide-reactive phosphine probe, rho-TEO-phosphine (21) (rho: rhodamine), has been designed and applied to attach a chromophore to azido-(K1)RNase A via Staudinger ligation at physiological pH indicating that the introduced azido group is accessible and could be addressed by other established azide-reactive bioorthogonal reaction schemes.
We describe here the development of a photoreleasable
version of
a protein phosphatase-1 (PP1)-disrupting peptide (PDP-Nal) that triggers protein phosphatase-1 activity. PDP-Nal is a 23 mer that binds to PP1 through several interactions. It was
photocaged on a tyrosine residue, which required the exchange of phenylalanine
in PDP-Nal to tyrosine in order to disrupt the most
important binding interface. This PDP-caged can
be light-controlled in live cells.
In this work, through a docking analysis of compounds from the ZINC chemical library on human β-tubulin using high performance computer cluster, we report new polycyclic aromatic compounds that bind with high energy on the colchicine binding site of β-tubulin, suggesting three new key amino acids. However, molecular dynamic analysis showed low stability in the interaction between ligand and receptor. Results were confirmed experimentally in in vitro and in vivo models that suggest that molecular dynamics simulation is the best option to find new potential β-tubulin inhibitors. Graphical abstract Bennett's acceptance ratio (BAR) method.
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