Three-dimensional electrophoretic NMR correlation spectroscopy for simultaneous structure determination of co-existing protein conformations

A novel method of three-dimensional electrophoretic NMR correlation spectroscopy (3D EP-COSY) has been proposed, developed, and implemented, It has a demonstrated potential of facilitating simultaneous structural assignments of multiple proteins in mixtures. The principle is to add a pulsed DC electric field that introduces a new dimension of electrophoretic flow, in which resonances of different molecules can be separated by their electrophoretic migration rates. As a result, two COSY spectra were simultaneously obtained in a single 3D EP-COSY experiment from a mixture of 150 mM L-aspartic acid and 148 mM 4,9-dioxa-1,12-dodecanediamine, with concurrent resolution of their chemical shifts and J-coupling constants. This novel approach creates a new horizon of multi-dimensional electrophoretic NMR. The technical break-through opens doors for structure characterization of complex protein systems and protein interactions, which are at the basis of biochemical mechanisms and phenomena of living systems.