Analysis tools for single-monomer measurements of self-assembly processes

Hoyer, Maria; Crevenna, Alvaro H.; Kitel, Radoslaw; Willems, Kherim; Czub, Miroslawa; Dubin, Grzegorz; Van Dorpe, Pol; Holak, Tad A. und Lamb, Don C. (18. März 2022): Analysis tools for single-monomer measurements of self-assembly processes. In: Scientific Reports, Bd. 12

Abstract

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for Spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin.

• Dokumententyp: Zeitschriftenartikel
• Publikationsform: Publisher's Version
• Fakultät: Chemie und Pharmazie > Department Chemie
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie
• ISSN: 2045-2322
• Sprache: Englisch
• DFG: Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 491502892