Nanoplasmonic sensing of multi-molecular protein interactions at physiological conditions

Projektinformationen

MultiSense

ID Finanzhilfevereinbarung: 864772

DOI

10.3030/864772

EK-Unterschriftsdatum 28 Januar 2020

Startdatum 1 September 2020

Enddatum 31 Dezember 2027

Finanziert unter

• EXCELLENT SCIENCE - European Research Council (ERC)

Gesamtkosten

€ 1 999 687,00

EU-Beitrag

€ 1 999 687,00

Koordiniert durch

TECHNISCHE UNIVERSITEIT EINDHOVEN
Netherlands

Periodic Reporting for period 3 - MultiSense (Nanoplasmonic sensing of multi-molecular protein interactions at physiological conditions)

Berichtszeitraum: 2023-09-01 bis 2025-02-28

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

I will develop a single-molecule sensor that reveals multi-protein dynamics at micromolar concentrations to provide new understanding of how protein machinery functions in real-time. Proteins and their interactions are the cornerstone of biological processes. The dynamic cooperation between multiple species is key to most processes including chaperone-mediated protein folding, signal transduction, and metabolism. The dynamics of these processes is fast and adaptive due to a tailored combination of low affinity and high concentration. Current single-molecule sensors cannot capture these dynamics because (1) they only work in dilute solutions which perturbs the dynamics or (2) they only resolve a single species. Capturing dynamics of protein machinery at physiological conditions therefore remains one of the grand challenges in the field.

MultiSense will develop a nanoplasmonic sensor to provide the opportunity to reveal multi-molecular protein dynamics at micromolar concentrations. This will be achieved by (a) developing technology to resolve and interpret multi-protein interactions and cooperation, and (b) using this technology to provide the first real-time picture of chaperone-mediated protein folding at physiological conditions. This will contribute to unraveling why chaperones fail to induce proper folding or prevent protein aggregation in the context of diseases.

The proposed method can be implemented on any research-grade microscope and can be generalized to any protein by applying the proper particle functionalization. This will inspire other researchers to study dynamic cooperation in protein machinery to unravel complex molecular mechanisms. In the long term the small size and biocompatibility of metal nanoparticles will enable studies of protein interactions at the single-molecule level in their natural environment, a living cell.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

The researchers have constructed the optical microscopy platform to perform multi-color single-molecule fluorescence measurements using plasmonic nanoparticles as signal enhancers. This platform is currently being expanded further to initiate and measure controllable temperature rises on the surface of nanoparticles. This will constitute a unique platform that enables simultaneous single-particle nanothermometry and single-molecule fluorescence experiments.

The necessary proteins and DNA constructs have been synthesized and functionalized with fluorophores to enable single-molecule fluorescence experiments of the chaperone cycle. Current work focuses on the optimization and quantification of protein- and peptide loading and accessibility on nanoparticle surfaces. Several publications have been written and will be submitted soon on the effect of particle coating on single-molecule interaction dynamics, the conjugation of low-QY dyes for single-molecule studies at high concentrations, and the numerical modelling and interpretation of pFRET signals.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

Already, the researchers have established a unique platform that enables simultaneous single-particle nanothermometry and single-molecule fluorescence experiments. This platform was used for multi-color single-molecule plasmon-enhanced fluorescence microscopy, and will in the coming period be expanded to enable single-molecule Forster Resonance Energy Transfer measurements. In the second phase of the project MultiSense will use this platform to reveal multi-molecular protein dynamics at micromolar concentrations and at controlled temperatures. This will provide the first real-time picture of chaperone-mediated protein folding at physiological conditions, and will contribute to unraveling why chaperones fail to induce proper folding or prevent protein aggregation in the context of diseases.

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