Studying protein dynamics with X-ray free-electron lasers: Opportunities & Limitations

Protein structure and function are intimately connected. To deduce the mechanisms underlying specific functions, it is therefore of high interest to investigate structural changes during a reaction. Recently, the development of serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) has attracted a great deal of attention by enabling time-resolved (TR) experiments at atomic spatial and femtosecond temporal resolution, thereby allowing unprecedented insight into protein dynamics. The high intensity of the XFEL pulse destroys any sample that has been exposed to the focused beam. A new protein crystal thus needs to be supplied for each pulse. This is typically achieved using a continuously flowing jet. For light-triggered reactions, an optical pulse starts the reaction in crystals of photosensitive proteins and the X-ray pulse then interrogates the system after a given time interval. For such experiments there are two main issues: First, appropriate conditions have to be found for triggering the reaction of interest. Second, the measurement of weak signals is severely limited by the low data collection rate (≤ 120 Hz) at first-generation XFELs. Moreover high sample consumption is an issue at these X-ray sources. The goals of this thesis were therefore twofold: In the first part, techniques were developed to enable studying the ultrafast isomerization following photon absorption by bacteriorhodopsin in a TR-SFX experiment. Extending these results, light-matter interactions changing the incident excitation intensity were quantified based on experiments and calculations. This allowed establishing guidelines how to generally determine appropriate excitation conditions in SFX employing light triggering. These findings are fundamental to avoid multiphoton artefacts arising from excessive excitation and are thus essential for studying biological reactions which take place almost exclusively in the single photon regime. In the second part of this thesis, opportunities and challenges of SFX experiments at next-generation XFELs were explored. These new machines generate X-ray pulses at MHz peak repetition rate and promise significantly higher throughput and more efficient sample usage. However, the short spacing between pulses introduces new challenges: it needs to be ensured that fresh sample is supplied sufficiently fast for each X-ray pulse. Moreover, it has been shown that the XFEL pulse launches shock waves in the sample carrying jet. These may damage sample probed by subsequent pulses. Here, first experiments at MHz peak repetition rate were conducted to investigate both issues. It was demonstrated that data collection of undamaged sample is indeed possible at 1.1 MHz repetition rate. At shorter pulse intervals (corresponding to 4.5 and 9.2 MHz), shock wave induced damage may lead to a significant loss in diffraction resolution of the crystal and even to structural changes in the protein. Together, the results of this thesis delineate the limitations of (TR-) SFX due to XFEL induced shock damage and pave the way towards exploiting the promising capabilities of MHz XFELs, in particular for studying biologically relevant light-triggered reactions in proteins.Proteinstruktur und –funktion sind eng miteinander verbunden. Um die zugrundeliegenden Mechanismen aufzuklären, ist es daher von hohem Interesse, strukturelle Änderungen während einer Reaktion zu verfolgen. Die Entwicklung serieller Femtosekunden-Kristallographie (SFX) an Freie-Elektronen-Lasern im Röntgenbereich (XFEL) hat folglich durch die einmalige Kombination von atomarer räumlicher und Femtosekunden zeitlicher Auflösung viel Aufmerksamkeit erregt, da sie beispiellose Einblicke in die Struktur und Dynamik von Proteinen erlaubt. XFEL Pulse besitzen eine solch hohe Intensität, dass die Probe letztendlich zerstört und für jeden Puls ein neuer Proteinkristall benötigt wird. Ein Flüssigkeitsstrahl (Jet) liefert daher kontinuierlich frisches Material. Mit diesem Ansatz lassen sich auch lichtgesteuerte Reaktionen beobachten, indem ein optischer Puls die Reaktion in einem Kristall aus photosensitiven Proteinen startet, und der Röntgenpuls nach einer festgelegten Zeit das System abfragt. Bei dieser Herangehensweise gibt es zwei grundlegende Probleme: Erstens müssen geeignete Bedingungen zum Starten der Reaktion gefunden werden. Zweitens ist an XFELs der ersten Generation die Messung schwacher Signale durch die geringe Repetitionsrate (≤ 120 Hz) limitiert, die zudem zu einem hohen Probenverbrauch führt. Diese Arbeit hat daher zwei Ziele: Im ersten Teil wurden Methoden entwickelt, die die Grundlage für das Verfolgen der ultraschnellen lichtinduzierten Isomerisierung in Bacteriorhodopsin mittels SFX bildet. Anknüpfend daran wurden die Anregungsintensität ändernde Licht-Materie-Wechselwirkungen mithilfe von Experimenten und Berechnungen quantifiziert, sodass ein allgemeiner Leitfaden für die Bestimmung passender Anregungsbedingungen aufgestellt werden konnte. Dies ist ein entscheidender Schritt für das Vermeiden biologisch irrelevanter Multiphotonen-Effekte. Im zweiten Teil der Arbeit wurden die Chancen und Herausforderungen von SFX an neuen XFELs untersucht, die Röntgenpulse mit bis zu MHz Wiederholrate produzieren können und dadurch versprechen, Durchsatz und Probeneffizienz zu erhöhen. Durch die kurzen Pulsabstände entstehen jedoch neue Probleme: einerseits muss die Zufuhr neuer Kristalle in den Strahl schnell genug geschehen. Andererseits wurde gezeigt, dass der XFEL Puls im Jet Schockwellen auslöst, die die Probe schädigen und so die Messung mit schnell aufeinanderfolgenden Pulsen beeinträchtigen könnte. In dieser Arbeit wurden erste Experimente bei MHz Wiederholrate durchgeführt und beide Problematiken untersucht. Messungen bei 1.1 MHz konnten erfolgreich ohne Beeinträchtigung durchgeführt werden. Es wurde aber auch gezeigt, dass bei kürzeren Pulsintervallen (entsprechend 4.5 und 9.2 MHz) die Schockwelle die Probe schädigen kann und dadurch zu einer reduzierten Auflösung der Kristalle, sowie zu Strukturänderungen im Protein führen können. Die Ergebnisse dieser Arbeit sind wegweisend für das Ausschöpfen der vielversprechenden Möglichkeiten von MHz XFELs, insbesondere für das Beobachten biologisch relevanter, ultraschneller, lichtinduzierter Reaktionen in Proteinen

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins

Viscous hydrophilic injection matrices for serial crystallography

Serial (femtosecond) crystallography at synchrotron and X-ray free-electron laser (XFEL) sources distributes the absorbed radiation dose over all crystals used for data collection and therefore allows measurement of radiation damage prone systems, including the use of microcrystals for room-temperature measurements. Serial crystallography relies on fast and efficient exchange of crystals upon X-ray exposure, which can be achieved using a variety of methods, including various injection techniques. The latter vary significantly in their flow rates – gas dynamic virtual nozzle based injectors provide very thin fast-flowing jets, whereas high-viscosity extrusion injectors produce much thicker streams with flow rates two to three orders of magnitude lower. High-viscosity extrusion results in much lower sample consumption, as its sample delivery speed is commensurate both with typical XFEL repetition rates and with data acquisition rates at synchrotron sources. An obvious viscous injection medium is lipidic cubic phase (LCP) as it is used for in meso membrane protein crystallization. However, LCP has limited compatibility with many crystallization conditions. While a few other viscous media have been described in the literature, there is an ongoing need to identify additional injection media for crystal embedding. Critical attributes are reliable injection properties and a broad chemical compatibility to accommodate samples as heterogeneous and sensitive as protein crystals. Here, the use of two novel hydro­gels as viscous injection matrices is described, namely sodium carb­oxy­methyl cellulose and the thermo-reversible block polymer Pluronic F-127. Both are compatible with various crystallization conditions and yield acceptable X-ray background. The stability and velocity of the extruded stream were also analysed and the dependence of the stream velocity on the flow rate was measured. In contrast with previously characterized injection media, both new matrices afford very stable adjustable streams suitable for time-resolved measurements

Influence of pump laser fluence on ultrafast structural changes in myoglobin

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer 1 . However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore 2-14 . As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern 15 whether this experimental approach 16 allows valid inferences to be drawn vis-à-vis biologically relevant single-photon-induced reactions 17 . Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics 15 ) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments 16 such that biologically relevant insight emerges

Shock Damage Analysis in Serial Femtosecond Crystallography Data Collected at MHz X-ray Free-Electron Lasers

International audienceSerial femtosecond crystallography (SFX) data were recorded at the European X-ray free-electron laser facility (EuXFEL) with protein microcrystals delivered via a microscopic liquid jet. An XFEL beam striking such a jet may launch supersonic shock waves up the jet, compromising the oncoming sample. To investigate this efficiently, we employed a novel XFEL pulse pattern to nominally expose the sample to between zero and four shock waves before being probed. Analyzing hit rate, indexing rate, and resolution for diffraction data recorded at MHz pulse rates, we found no evidence of damage. Notably, however, this conclusion could only be drawn after careful identification and assimilation of numerous interrelated experimental factors, which we describe in detail. Failure to do so would have led to an erroneous conclusion. Femtosecond photography of the sample-carrying jet revealed critically different jet behavior from that of all homogeneous liquid jets studied to date in this manner

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin

International audienceBacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event uponlight absorption is isomerization of the retinal chromophore. Here we used time-resolvedcrystallography at an X-ray free-electron laser to follow the structural changes inmultiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry andultrafast spectroscopy were used to identify a sequential two-photon absorption process,leading to excitation of a tryptophan residueflanking the retinal chromophore, as afirstmanifestation of multiphoton effects. We resolve distinct stages in the structural dynamics ofthe all-transretinal in photoexcited bR to a highly twisted 13-cisconformation. Other activesite sub-picosecond rearrangements include correlated vibrational motions of the electro-nically excited retinal chromophore, the surrounding amino acids and water molecules as wellas their hydrogen bonding network. These results show that this extended photo-activenetwork forms an electronically and vibrationally coupled system in bR, and most likely in allretinal proteins

Influence of pump laser fluence on ultrafast myoglobin structural dynamics

International audienceHigh-intensity femtosecond pulses from an X-ray free-electron laser enable pump–probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer 1,2 . However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore 3–17 . As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern 18,19 whether this experimental approach 20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions 18,19 . Here we describe ultrafast pump–probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe–CO bond distance (predicted by recent quantum wavepacket dynamics 21 ) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump–probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump–probe experiments 20 such that mechanistically relevant insight emerges

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan , respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo -grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control

MHz data collection of a microcrystalline mixture of different jack bean proteins

Design Type(s)protocol testing objectiveMeasurement Type(s)protein structure dataTechnology Type(s)x ray crystallographyFactor Type(s)Sample Characteristic(s)Canavalia Machine-accessible metadata file describing the reported data (ISA-Tab format