A new route to protein chemistry using high-speed atomic force microscopy

Datum

15.01.2019

Zeit

11:00 - 12:00

Sprecher

Adrian Nievergelt

Zugehörigkeit

EPFL, Switzerland

Sprache

en

Hauptthema

Biologie

Andere Themen

Biologie

Host

Gaia Pigino

Beschreibung

Atomic force microscopy has long since been established as a powerful method in molecular biology. The invention of high-speed atomic force microscopy has been a key enabling technology for studying the dynamics of small biomolecules, and especially proteins at the single molecule level. Among such dynamic effects, the biogenesis of individual proteins into complex structures is one of significant interest. Many of these complexes, for example microtubules, need to be assembled and disassembled over time in order to fulfill their physiological role. As strongly bound structures require a significant energy investment to disassemble, such dynamic nature is frequently linked to low binding energies. One striking example of such a weakly-bound protein is SAS-6, a building block that assembles into nine-fold symmetric rings that ultimately act as a template for centriole formation in eukaryotic cells. However, imaging of the ring formation process by atomic force microscopy requires both fast imaging speeds of only a few seconds per frame, as well as probing forces that are currently not obtainable with state of the art techniques. Recent developments in instrumentation which are allowing cleaner probe excitation such as photothermal drive allow for an increased robustness of measurements as well as a significant increase in achievable imaging speed. Our open-source high-speed atomic force microscopy platform has enabled us the development of a novel measurement technique with probing forces that are unprecedentedly low in high-speed atomic force microscopy. Using laser-based thermal actuation, combined with high-speed time-domain analysis of the resulting probing motion, we are able to record the fragile formation process of full SAS-6 rings at nanometer resolution. The resulting recordings show assembly pathways from single elements to 9-fold symmetric rings, as well as for the first time give an estimate for the ring formation time in-vivo. Finally, using computer vision, we extract from our high-speed AFM data otherwise inaccessible surface chemical properties such as surface diffusion, local concentrations and the surface association and disassociation rates.

Letztmalig verändert: 16.01.2019, 01:08:02