Community Research and Development Information Service - CORDIS

Periodic Report Summary 1 - PROTEINFED (Towards Femtosecond Electron Diffraction of Proteins)

Summary description of the project objectives
The aim of this project is to extend the application of the newly developed technique femtosecond
electron diffraction (FED), a method to measure ultrafast structural dynamics, to proteins to gain insight
into protein motions in real-time with up to atomic spatial resolution. Special focus for the time spent with the
outgoing host is on sample development using Bacteriorhodopsin (BR) and Myoglobin/Hemoglobin
(Mb/Hb) as ideal proteins for technology development, ultrafast electron diffraction measurements are planned for the return phase with the host. The scientific question driving the project is to develop an experimental tool to study protein dynamics with full atomic resolution on the most fundamental chemical timescale starting in the femtosecond regime. This is a research topic of increasing interest, as it will allow to understand the fundamental basis of biological function including processes such as signal transduction or enzymatic reactions.

The actual work objectives for this project during the outgoing phase, carried out at University of Toronto, Canada, were the testing of different approaches for sample preparation, further sample delivery as well as first electron diffraction tests for the model proteins, in the return phase time-resolved electron diffraction shall be performed.

Work performed since the beginning of the project and main results
In the outgoing period the first protein samples for ultrafast electron diffraction were prepared and tested. The main model proteins investigated are Bacteriorhodopsin and Myoglobin. Bacteriorhodopsin is a proteocaryotic membrane protein with a structure similar to rhodopsin, itself a major example of a G-Protein coupled receptor (GPCR). GPCRs are of significant importance for pharmacological and biomedical applications as they are the largest protein family addressed by medications. Myoglobin is a well studied globular protein, which binds di-atomic ligands, like oxygen or carbonmonoxide.

The researcher learned techniques for protein crystallisation, including batch crystallisation and the approach of crystallizing membrane proteins in the lipidic cubic phase in the laboratory of Oliver Ernst, Department of Biochemistry at University of Toronto. Further she learnt and performed ultrafast electron diffraction experiments on a 95keV machine in the physics department at University of Toronto. Additionally time-resolved experiments using X-Rays as structural probe (time-resolved serial femtosecond crystallography) have been carried out at both X-Ray Free Electron Lasers (XFELs) running worldwide, with one experiment at LCLS/US in March 2015 and one at SACLA/Japan in April 2016. These experiments were focused on sample of Myoglobin with extension to retinalproteins.

Main results achieved so far
From the crystallization trials first samples for testing electron diffraction on proteins were derived (no further details given in this summary as results are not published yet). Further a fixed target for sample delivery for serial crystallography has been tested in XFEL experiments, its application will be expanded for electron experiments. Significant results on the ultrafast dynamics upon ligand dissociation in myoglobin on the 100 fs to 2ps timescale under low excitation conditions have been found.

Expected final results and their potential impact and use
At the end of the project it is envisioned that a clear method for static and time-resolved structure determination of proteins using ultrafast electron diffraction is established or in case of major problems at least all technical constraints are known and solutions for this are known. The understanding of ultrafast protein dynamics by studying the motions with full atomic detail starting on the femtosecond timescale will be subject of applying the methods developed throughout this project. The expected impact is of major importance, as this will allow a completely new route for structural biology and give access to study protein function in all detail in a table-top experiment.

Contact

RJ Dwayne Miller, (Director)
Tel.: +123456789
E-mail

Subjects

Life Sciences
Record Number: 187593 / Last updated on: 2016-08-22
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