We have developed a novel approach to elucidate the structure and transient behavior of protein molecules at the solution-solid interface based on dynamic single-molecule tracking. This approach, which is uniquely sensitive to structural and interfacial dynamics, includes the use of high-throughput tracking of protein molecules by means of internal reflection fluorescence (TIRF) microscopy in combination with intramolecular as well as intermolecular Forster resonance energy transfer (FRET). An important aspect of this approach is the use of bioothogonal labeling techniques to site-specifically introduce donor and acceptor fluorophores, which allow fluctuations in FRET efficiency to be correlated with changes in protein structure. Notably, in this approach, as many as 10<sup>6</sup> protein molecules are tracked as they adsorb, desorb, diffuse, and simultaneously undergo conformational changes and/or intermolecular associations, permitting the statistical identification of dynamic, spatial, and population heterogeneity. The subsequent correlation of these dynamic behaviors on a molecule-by-molecule basis via large-scale multi-variate analyses, moreover, provides new insights into the connection between interfacial dynamics and protein structure. This talk will specifically focus on the application of this approach to understand the connection between protein dynamics and conformation as well as function.

Connecting protein structure, function, and interfacial dynamics with single-molecule methods

255th National Meeting (2018)

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