2D-measurement of proton T1ρ relaxation in unlabeled proteins: Mobility changes in α-bungarotoxin upon binding of an acetylcholine receptor peptide.

摘要

A method for the measurement of proton T1ρ relaxation times in unlabeled proteins is described using a variable spin-lock pulse after the initial non-selective 90° excitation in a HOHAHA pulse-sequence. The experiment is applied to α-bungarotoxin (α-BTX) and its complex with a 25-residue peptide derived from the acetylcholine receptor (AChR) α-subunit. A good correlation between high T1ρ values and increased local motion is revealed. In the free form, toxin residues associated with receptor binding according to the NMR structure of α-BTX complex with an AChR peptide and the model for the α-BTX with the AChR display high mobility. Upon binding the AChR peptide a decrease in the relaxation times and motion of residues involved in binding of the receptor α-subunit is exhibited, while residues implicated in binding γ and δ subunits retain their mobility. In addition, the quantitative T1ρ measurements enables us to corroborate the mapping of boundaries of the AChR determinant strongly interacting with the toxin and can similarly be applied to other protein complexes in which peptides represent one of the two interacting proteins. The presented method is advantageous due to its simplicity, generality and time efficiency and paves the way for future investigation of proton relaxation rates in small unlabeled proteins.