Protein Sizing and Conformation Analysis with an Electro-Switchable DNA Chip

Abstract

Powerful methods for analyzing proteins and their interactions with small molecules, nucleic acids or other proteins are a cornerstone in the development of new drugs and next-generation disease diagnostics. Commonly, affinity and kinetics of molecular interactions are determined with surface biosensors, which measure the adsorption/desorption of solute target molecules to/from surface immobilized capture molecules. However, information on protein size, shape (folding state), molecular aggregates, or chemically subtle changes such as post-translational modifications, usually remain unrevealed and must be identified in complementary assays.Here, we show that by analyzing the molecular dynamics of proteins, set in motion by electrically actuated DNA ‘levers’, it is possible to measure protein size and detect changes in protein structure. Simultaneously, chemical rate constants and dissociation constants are obtained. The method is label-free, uses a parallel microelectrode format for multiplexed assays and microfluidics for low sample consumption. An analytical model based on continuum electrostatic Poisson-Boltzmann theory and the Fokker-Planck equation allows to determine the protein's diameter with sub-nanometer accuracy. Evaluating an extensive set of molecular dynamics data with ligand proteins of differing size, we find excellent agreement between theory and experiment.The devised concepts open up a novel route for protein analysis on a chip. The relative abundance of antibody fragments in heterogeneous mixtures can be determined, the unfolding of the protein tertiary structure is easily detected and even the ion-induced conformational change of a protein is observed in real-time. Moreover, we demonstrate the revelation of post-translational modifications at the example of a glycosylation and a phosphorylation.Due to the unprecedented information content about the investigated proteins, offered by this novel technology, we believe that we can provide an essential prerequisite for the progress in early identification of promising leads when screening for biological drug candidates.