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Tracking the structural dynamics of ligand-protein interactions using X-ray free electron lasers

English title Tracking the structural dynamics of ligand-protein interactions using X-ray free electron lasers
Applicant Standfuss Joerg
Number 179351
Funding scheme Project funding (Div. I-III)
Research institution Paul Scherrer Institut
Institution of higher education Paul Scherrer Institute - PSI
Main discipline Biophysics
Start/End 01.05.2018 - 30.04.2022
Approved amount 904'000.00
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All Disciplines (4)

Discipline
Biophysics
Molecular Biology
Structural Research
Pharmacology, Pharmacy

Keywords (8)

retinal-binding proteins; G protein-coupled receptors; rhodopsins; serial femtosecond crystallography; chemokine receptor; X-ray free electron lasers; time-resolved serial crystallography; photopharmacology

Lay Summary (German)

Lead
In 2017 wurde am Paul Scherrer Institut eine neue Grossforschungsanlage, der Schweizer Freie-Elektronen Laser (SwissFEL), in Betrieb gestellt. Diese Anlage ist ideal um sehr schnell ablaufende biologische Reaktionen in Proteinmolekülen zu untersuchen. Im gewissen Sinne arbeitet der SwissFEL wie eine Kamera mit der mehrere Schnappschüsse solcher Reaktionen aufgenommen und anschliessend zu einem Film der Reaktion zusammengefügt werden können. Solche Filme werden uns helfen die Wirkungsweise der Proteine besser zu verstehen. Insbesondere für die pharmazeutische Industrie sind Proteine aufgrund ihrer Schlüsselfunktionen ein begehrtes Forschungsobjekt.
Lay summary

In diesem Projekt werden wir die Funktionsweise einer Reihe von Proteinen mittels des SwissFELs und ähnlicher Anlagen in den USA und Japan untersuchen. Unser Fokus liegt dabei zunächst auf von Licht aktivierbaren Proteinen die mittels des Sehfarbstoffes Retinal angetrieben werden. Zu diesen Protein gehört z.B. das visuelle Rhodopsin in unserem Auge, aber auch bakterielle lichtgetriebene Ionenpumpen die zur Photosynthese beitragen.

In einem zweiten Schritt werden wir unsere Forschungen auf pharmakologisch wichtige Proteine wie einem humanen Chemokine Rezeptor und dem wichtigen Zellbaustein Tubulin ausweiten. Chemokine Rezeptoren sorgen in unserem Immunsystem dafür, dass unsere Abwehrzellen dorthin wandern, wo sie ungeliebte Eindringlinge wie Viren oder Bakterien am effektivsten bekämpfen können. Tubulin ist an der Zellteilung beteiligt und ein viel genutzter Angriffspunkt für die chemotherapeutische Behandlung von schnellwachsenden Tumorzellen. Die moderne Medikamentenforschung setzt voraus, dass man die Strukturen der Proteintargets und ihrer Verbindungen mit potenziellen neuen Medikamenten kennt. Wir erhoffen uns, dass unsere Forschung hier neue Möglichkeiten eröffnen wird, weil sie zum Ziel hat neben der Struktur auch die Bewegung der Medikamente innerhalb der Zielproteine sichtbar zu machen.

Direct link to Lay Summary Last update: 28.05.2018

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
159558 Structural dynamics of 7TM proteins probed by serial femtosecond crystallography 01.05.2015 Project funding (Div. I-III)
177125 Tunable nanosecond laser for time-resolved serial crystallography at SLS and SwissFEL 01.01.2018 R'EQUIP

Abstract

This proposal addresses one of the major promises of X-ray free electron laser (XFEL) technology: to advance structural biology from the determination of static structures to dynamic molecular movies. We will use this cutting-edge technology to study the dynamics of ligand-protein interactions with the temporal precision of ultrafast spectroscopy and the near atomic resolution of X-ray crystallography.In the last few years we have established serial crystallography at the Swiss Light Source (SLS) and studied the light-driven proton pump bacteriorhodopsin (bR) using time-resolved serial femtosecond crystallography (TR-SFX) at X-ray lasers. Temporal snapshots ranging from femtoseconds to milliseconds provide unique molecular insights into the photocycle and the initial photochemical reaction driving this prototypical retinal binding protein. Results have direct relevance to other retinal-binding proteins involved in maintaining the selectivity of biological membranes or human biological processes like our visual sense.In the current proposal, we suggest to build on these successes to study new protein targets using the natural retinal and synthetic azobenzene based photoswitches. Naturally light-sensitive proteins have paved the way for time-resolved studies using XFELs but these proteins correspond to only a small subset of biological systems. Harnessing the chemistry of synthetic photoswitches to study proteins that cannot be natively activated by light will dramatically increase the number of biological systems whose structural dynamics can be studied at modern XFEL sources. The Paul Scherrer Institute (PSI) provides an ideal research environment for the proposed research with access to well-equipped laboratory space in the Laboratory of Biomolecular Research (LBR), the Swiss Light Source (SLS) and the Swiss Free Electron Laser (SwissFEL). A close collaboration with medicinal chemists from the PSI spin-off company leadXpro will provide further multidisciplinary and translational aspects to the research. Initially, we will use a combination of TR-SFX and molecular dynamic simulations to characterize the conformational changes upon activation of retinal proteins. We will conclude our study of the bR photocylce and then enter more application focused research by expanding these studies to the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 (KR2). Understanding how light regulates the flow of ions across biological membranes has important implications for the optogenetic manipulation of neural cells. Synthetic photoswitches will mark the next step in our push from basic photoreceptor research towards a broader range of targets with a close focus on applications. The field of photopharmacology utilizes such light-sensitive compounds to activate and deactivate medicinal compounds by light. As an example, for the use of synthetic photoswitches in time-resolved crystallography, we will track dissociation of azo-combretastatin from / tubulin, the principal building block of the microtubule cytoskeleton. Microtubules have pivotal roles in cell division and survival and tubulin targeting agents are potent drugs widely used in chemotherapy to treat a variety of cancers. Finally, we will characterize new synthetic photoswitches to study G protein-coupled receptors (GPCRs), in particular the A2a adenosine and CCR7 chemokine receptors systems. Our aim is to use photodestructible or photoswitchable compounds to follow ligand unbinding and determine ligand-free structures of these receptors. So far, these states could not be crystallized because tightly bound ligands are needed to reduce flexibility. Yet the unliganded basal state is the critical starting point for GPCR function and medicinal intervention. Studying the dynamics of ligand recognition and dissociation with the near atomic spatial and femtosecond temporal precision of TR-SFX will thus provide invaluable insights into the molecular basis of pharmacology.
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