Presence Of Monophosphate Research Articles

Protein lysine-Nζ alkylation andO-phosphorylation mediated by DTT-generated reactive oxygen species

Reactive oxygen species (ROS) play crucial roles in physiology and pathology. In this report, we use NMR spectroscopy and mass spectrometry (MS) to demonstrate that proteins (galectin-1, ubiquitin, RNase, cytochrome c, myoglobin, and lysozyme) under reducing conditions with dithiothreitol (DTT) become alkylated at lysine-N(ζ) groups and O-phosphorylated at serine and threonine residues. These adduction reactions only occur in the presence of monophosphate, potassium, trace metals Fe/Cu, and oxygen, and are promoted by reactive oxygen species (ROS) generated via DTT oxidation. Superoxide mediates the chemistry, because superoxide dismutase inhibits the reaction, and hydroxyl and phosphoryl radicals are also likely involved. While lysine alkylation accounts for most of the adduction, low levels of phosphorylation are also observed at some serine and threonine residues, as determined by western blotting and MS fingerprinting. The adducted alkyl group is found to be a fragment of DTT that forms a Schiff base at lysine N(ζ) groups. Although its exact chemical structure remains unknown, the DTT fragment includes a SH group and a --CHOH--CH₂-- group. Chemical adduction appears to be promoted in the context of a well-folded protein, because some adducted sites in the proteins studied are considerably more reactive than others and the reaction occurs to a lesser extent with shorter, unfolded peptides and not at all with small organic molecules. A structural signature involving clusters of positively charged and other polar groups appears to facilitate the reaction. Overall, our findings demonstrate a novel reaction for DTT-mediated ROS chemistry with proteins.

Conformation of a pentacosapeptide representing the RNA-binding N-terminus of cowpea chlorotic mottle virus coat protein in the presence of oligophosphates: a two-dimensional proton nuclear magnetic resonance and distance geometry study.

Conformational studies were performed on a synthetic pentacosapeptide representing the RNA-binding N-terminal region of the coat protein of cowpea chlorotic mottle virus. Two-dimensional proton NMR experiments were performed on the highly positively charged peptide containing six arginines and three lysines in the presence of an excess of monophosphates, tetra(poly)phosphates, or octadeca(poly)phosphates mimicking the phosphates of the RNA. The results show that the peptide alternates between various extended and helical structures in the presence of monophosphate and that this equilibrium shifts toward the helical structures (with the helical region situated between residues 10 and 20) in the presence of oligophosphates. Distance geometry calculations using distance constraints derived from a NOESY spectrum of the peptide in the presence of tetra(poly)phosphate resulted in eight structures belonging to two structure families. The first family consists of five structures with an alpha-helixlike conformation in the middle of the peptide, and the second family consists of three structures with a more open conformation. The propensity to form an alpha-helical conformation in the N-terminal part of this viral coat protein upon binding of phosphate groups to the positively charged side chains is suggested to play an essential role in RNA binding.