Correlative smFRET/electrophysiology for long-term studies of protein dynamics

Información del proyecto

COSMO

Identificador del acuerdo de subvención: 101033291

DOI

10.3030/101033291

Proyecto finalizado el 12 Enero 2023

Fecha de la firma de la CE 12 Abril 2021

Fecha de inicio 13 Abril 2021

Fecha de finalización 12 Abril 2023

Financiado con arreglo a

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coste total

€ 166 320,00

Aportación de la UE

€ 166 320,00

Coordinado por

KATHOLIEKE UNIVERSITEIT LEUVEN
Belgium

Periodic Reporting for period 1 - COSMO (Correlative smFRET/electrophysiology for long-term studies of protein dynamics)

Período documentado: 2021-04-13 hasta 2023-04-12

Resumen del contexto y de los objetivos generales del proyecto

Proteins carry out complex biological processes that are essential for cell survival. Rather than being static, they are highly dynamic molecules whose function depends on their 3D structure and dynamic properties. The study of protein dynamics is not only of fundamental interest, but also has direct implications for health and disease, as altered dynamics can lead to disease. However, it remains difficult to experimentally determine the multitude of conformations adopted by proteins, their lifetimes and the transitions between them. Single molecule FRET (smFRET) is probably the most suitable technique available but is limited by the short observation times (a few seconds) and the difficulty of measuring more than one molecular distance at the same time.

The goal of this action “Correlative smFRET/electrophysiology for long-term studies of protein dynamics” is to develop an innovative correlative approach that can extend smFRET observations to much longer timescales, while also providing a way to integrate multiple measurements into a coherent molecular picture.

How is it done? by combining smFRET measurements with nanopore-based electrophysiological experiments that have high temporal resolution and are also sensitive to conformational changes. Our correlative system thus combines the speed and sensitivity of electrical reading with the direct observation of molecular distances using smFRET. This new methodology should open entirely new opportunities to monitor the fast and long-term conformational dynamics of proteins or the assembly processes of macromolecules.

This methodology is meant to map conformational dynamics of the biologically-important Ras proteins. Defects in Ras proteins are implicated in 30% of human cancers. We aim to better understand the conformational dynamics of selected Ras proteins with the ultimate goal of obtaining drugs that function through the regulation of these dynamics.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

The work was carried out through 3 work packages (WP) although there was no time left for the last one. The WP1 consisted in implementing the two techniques smFRET and electrophysiology based on nanopores in the host group with the aim of combining them on a microscope for simultaneous reading of the optical and electrical signal. New cameras, lasers and optics were required for smFRET experiments and results were obtained on immobilized proteins that exhibit two conformations. The electrophysiological experiments required many optimizations and took much longer than expected to obtain stable lipid bilayer droplets.

A major problem is the high salt concentrations needed for electrophysiology which are not compatible with the work of proteins. After many conditions tested, stable droplets were obtained consistently at low salt concentrations with nanopores insertions. Electrical traces of proteins trapped in the nanopores were recorded and, as expected, conformational changes were observed. In WP2, the two techniques were combined, which means that both signals were recorded simultaneously. Unfortunately, no FRET signal has yet been detected, only fluorescence. Calcium dyes and calcium biosensors have been studied by both electrophysiology and fluorescence.

Results for this action: The correlative set-up for optical imaging and electrophysiology is built and operational. Some small optimizations are still needed for the detection of the FRET signal but the fluorescence can already be detected. I believe detection of the smFRET signal is possible and will be done. Work is still in progress and has been presented at several conferences. Results are not yet available as open access publications. Protocols will also be made available.

The MSCA action also gives the possibility of sharing my passion for fluorescence microscopy with the general public with the organization of a Shine On exhibition! which has been on display in a public library for a few weeks. International researchers were invited for the opening ceremony and a symposium on related topics was organized.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

Once the smFRET signal can be detected simultaneously with electric current, it will be possible to study the conformational dynamics of proteins for a much longer period of time than is possible now with smFRET alone. This will allow accumulating more data on structural states and the transition sequence between them and perhaps seeing rarely populated states. This research is intended to be apply to a relevant class of proteins, the Ras proteins. As mentioned above, Ras gene mutations can ultimately lead to cancer. Our collaborator (Gouridis) showed that some of them have a more complex behavior than previously published (switching between ON and OFF states). We anticipate that identifying all conformational states of these proteins will likely to open up entirely new avenues for rational drug design.

Shine On Exposition.