Visualizing the Conformational Dynamics of Proteins by Time-Resolved Electron Microscopy

Projektinformationen

ProteinDynamics

ID Finanzhilfevereinbarung: 759145

DOI

10.3030/759145

Projekt abgeschlossen

EK-Unterschriftsdatum 30 August 2017

Startdatum 1 März 2018

Enddatum 31 August 2023

Finanziert unter

• EXCELLENT SCIENCE - European Research Council (ERC)

Gesamtkosten

€ 2 000 000,00

EU-Beitrag

€ 2 000 000,00

Koordiniert durch

ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE
Switzerland

Periodic Reporting for period 4 - ProteinDynamics (Visualizing the Conformational Dynamics of Proteins by Time-Resolved Electron Microscopy)

Berichtszeitraum: 2022-09-01 bis 2023-08-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Proteins make up the machinery of life and perform a multitude of tasks in living organisms. They are involved in harvesting energy from the environment, processing nutrients, sensing stimuli, or reproduction of the organism. Understanding the function of a protein may allow us to regulate it or combat dysfunction related to disease and is therefore crucial for biomedical applications. Much of our knowledge of proteins derives from atomic-resolution structures that are obtained with methods in structural biology, such as x-ray crystallography or cryo-electron microscopy. The structures such obtained are static, with available methods giving only limited information on the motions of proteins. However, by their very nature, proteins are dynamic systems, nanoscale machines that undergo a range of motions to perform their tasks. In the absence of direct observations of these dynamics, our understanding remains fundamentally incomplete. The goal of this project is to develop novel methods for imaging proteins while they perform their task, so as to obtain a movie of the processes involved. To give an example of a potential application – observing how the presence of a drug molecule alters the motions of a protein would significantly improve our understanding of the action of a drug and help researchers develop new types of medication.

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 approach we are pursuing for watching the motions of proteins in real time involves high-speed observations with an electron microscope. While electron microscopes excel at recording atomic-resolution images of proteins, they are currently not fast enough to capture most protein motions, which typically occur on a timescale of microseconds to milliseconds. We have developed a new approach that overcomes these limitations by developing a microsecond time-resolved variant of cryo-electron microscopy. In cry-electron microscopy, the three-dimensional structure of proteins in determined by imaging them with an electron microscope, with the proteins embedded in so-called vitreous ice. This is a glassy state of ice that can be produced by rapidly cooling a solution of the proteins, so that the solution does not have time to crystallize. We have shown that we can use a laser beam to rapidly melt such a cryo sample. Once the sample is liquid, we can induce dynamics of the embedded proteins. As they unfold, we then switch the laser off that keeps the sample liquid, so that it cools rapidly and revitrifies, trapping the proteins in their transient states, in which we can subsequently image them. Importantly we can do so rapidly, with microsecond time resolution. Moreover, we have shown that we can obtain near-atomic resolution structures of the proteins with this approach.

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)

Most recently, we have for the first time applied our approach to a real-life system, by studying the dynamics of the capsid of CCMV, a plant virus. Our work elucidates the intricate, microsecond motions of the capsid of the virus that are crucial for its viral life cycle and helps us understand the workings of this sophisticated nanoscale machine. Importantly, the methodology we have developed as part of the ERC grant is currently the only technique that allows one to observe these dynamics. Moreover, we believe that it will enable studies of the motions of a wide range of proteins. This promises to fundamentally advance our understanding of how proteins function.

Molecular models of the capsid of CCMV in its extended (left) and contracted state (right).