Direct, Single Molecule, Cell-by-Cell Observation of Molecular Kinetics and Thermodynamics in Early Lymphocyte Signaling

Abstract

stability of the employed oligonucleotide nanolevers under different electrolyte conditions. Temperature dependent data are presented from 4 to 70 C for a range of different bacterial and human proteins: while some show a straightforward unfolding transition, others feature more complex signatures which are indicative of the protein expansion before an unfolding of individual domains sets in at higher temperatures. We discuss the possibility to evaluate quantitative thermodynamic parameters (melting points, transition energies) from the molecular dynamics data and compare our results to established methods like thermofluor assays. Since the presented approach works on a chip surface it uses minimal amounts of sample and can be performed in parallel using a microelectrode array. In addition, it yields binding kinetics data (association and dissociation rates) and affinity values and thus is ideally suited for investigating interactions in combination with a thermodynamic characterization of the involved proteins.