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.
Item Type: | Journal article |
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Form of publication: | Publisher's Version |
Faculties: | Chemistry and Pharmacy > Department of Chemistry |
Subjects: | 500 Science > 540 Chemistry |
URN: | urn:nbn:de:bvb:19-epub-104685-8 |
ISSN: | 2045-2322 |
Language: | English |
Item ID: | 104685 |
Date Deposited: | 13. Jul 2023, 13:44 |
Last Modified: | 08. Apr 2024, 13:27 |
DFG: | Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 491502892 |