Measurement and analysis of the first assembly steps in protein polymerization

Proteins play a crucial role throughout all phyla of life. Many proteins build long filamentous structures by assembling many globular single proteins, the monomers. This process is both associated with physiological functions in the organism and with pathological malfunctions, resulting in disease. Although filament elongation has been extensively studied, the mechanism of filament nucleation remains often unclear. High background from the micromolar concentrations needed for filament formation have prevented direct observation of the nucleation dynamics using microscopy or spectroscopy methods. To directly monitor the early steps of filament assembly, I have used the attoliter excitation volume of zero-mode waveguides (ZMW). Thereby, a tethering protein on the bottom of the ZMW is used to bind the monomers, which start assembling after the start of the measurement. The resulting data is presented as a step-wise increase in intensity over time, each step indicating the binding of a monomer. Now that it is possible to obtain step-wise, single-filament data, adequate analysis methods need to be developed. Here, I present two new analysis methods for single-filament data, the visitation analysis and the average rate analysis. These methods, as well as the classic dwell-time analysis often used for the analysis of microscopic association and dissociation rates, have been tested and compared using simulations of filament nucleation. Furthermore, the limitations of each analysis method have been explored along the lines of the signal-to-noise ratio, the sampling rate, the labeling efficiency and the photobleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. The results of these simulations allow one to analyze and interpret single-filament data in a meaningful way and to plan the experimental conditions according to the kinetics and maximum labeling efficiency of the assembling protein of interest. The developed analysis methods have been applied to experimental data of actin nucleation. Using single tethering proteins attached to the bottom of the ZMW, the actin filament nucleation process could be visualized using different actin nucleators. For pointed-end actin growth on gelsolin, two distinct populations depending on the stability of the actin dimer were found, of which only one lead to stable filament elongation. Furthermore, barbedend actin growth was monitored using the nucleators cappuccino and spire. Whereas cappuccino was found to nucleate efficiently, leading to stable growing oligomers, spire was found to not be sufficient for actin nucleation. Using actin-binding compounds like Latrunculin A, which inhibits the flattening of the actin monomers occurring during filament formation, flattening was determined to be a key requirement for the formation of stable elongating filaments. Miuraenamide A, another actin-binding compound was found to stabilize oligomers and reduce the critical concentration needed for filament formation