Macromolecular interactions at the single-molecule level - Fluctuation analysis of bound states

The recognition, binding and release of macromolecules are precisely orchestrated in a stochastic interplay governing all enzymatic, immunological and cellular processes. Deciphering this complex synergy in order to understand these fundamental biological processes has been a matter of active research for decades; structural models and ensem- ble averaging methods continue to offer insight into the vivid nature of macromolecules. Though, just as the most gifted musician cannot tell the score of a single violin through a musical ensemble, the scientist cannot tell the trajectory of a single molecule by study- ing molecular ensembles. Thus, in order to characterize the true fluctuating dynamics of biological macromolecules, single-molecule sensitivity is a requirement. Here, a method for the extraction of single-molecule fluctuation parameters has been developed from the bottom up. The method combines total internal reflection fluo- rescent microscopy, surface immobilization, ligand-fluorescent polystyrene nanoparticle conjugation and video analysis to study the binding kinetics of macromolecular pairs. In short, videos of fluorescent nanoparticles were recorded using total internal reflection fluorescent microscopy, and their time-dependent movements were analyzed. Informa- tion on the retention times on the substrate was extracted and related to variations in the surface functionalization of both the nanoparticles and the substrate. As a part of this, an algorithm was developed and shown to reproduce published results from the literature and extracted correct kinetic parameters from a virtually generated image series. The capability of the assay was also demonstrated by characterizing the interaction between the surface-bound polysaccharide polymannuronan and nanopar- ticles functionalized with its C5 epimerase AlgE4. It was found that the fluorescent polystyrene nanoparticles were inapplicable due to high degrees of unspecific binding and reliance on electrostatic stabilization. However, it is expected that utilizing more dispersible fluorescent probes with a smaller fingerprint would allow the method to be applicable for a range of macromolecular pairs at physiological conditions.