Structure and dynamics of biomacromolecules in solution: recent developments and future perspectives in SANS/SAXS and neutron spectroscopy

This French University Habilitation (“mémoire” to obtain the “Diplôme d’Habilitation à Diriger des Recherches”, DHDR) is divided into two parts: the first one deals with the results that I have obtained after my PhD thesis in 2003, the second one discusses open questions related to theseresults as well as mid- and long-term perspectives.Three different topics are presented in the result chapters: 1) a combination of small angle scattering (SAS) and nuclear magnetic resonance (NMR) for rigid-body modeling of biomacromolecular complexes, 2) the combined use of small angle X-ray (SAXS) and neutron (SANS) scattering for the study of unfolded proteins and 3) the study of biomacromolecular and solvent dynamics by neutron spectroscopy combining several instruments.Points 1) and 3) are discussed in great detail, both in the results and perspective sections, since they represent the most advanced projects of my research. Point 2) is dealt with more briefly in the results section, since few results are available so far. However, perspectives are discussed. Aspecial perspective chapter deals with applications on membrane proteins. Key publications for the different chapters are:Chapter 1: Gabel et al. (2006) A target function for quaternary structural refinement from small angle scattering and NMR orientational restraints. Eur. Biophys. J. 35(4), 313-327. Gabel et al. (2008) A structure refinement protocol combining NMR residual dipolar couplings and small angle scattering restraints. J. Biomol. NMR 41(4), 199-208.Chapter 2: Gabel et al. (2009) Quantitative Modelfree Analysis of Urea Binding to Unfolded Ubiquitin Using a Combination of Small Angle X-ray and Neutron Scattering. J. Am. Chem. Soc. 131(25), 8769-8771.Chapter 3: Gabel (2005) Protein dynamics in solution and powder measured by incoherent elastic neutron scattering: the influence of Q-range and energy resolution. Eur. Biophys. J. 31(1), 1-12. Gabel & Bellissent-Funel (2007) C-Phycocyanin Hydration Water Dynamics in the Presence of Trehalose: An Incoherent Elastic Neutron Scattering Study at Different Energy Resolutions. Biophys. J. 92(11), 4054-4063.The habilitation thesis is focused on methodological aspects and developments of small angle scattering and neutron spectroscopy. It is obvious that the approaches discussed here and their sophisticated levels of data analysis rely fundamentally on the quality of the sample, and inparticular on monodispersity (for SAS) and amount of material for spectroscopy. The paramount importance of good biochemistry and the use of complementary techniques for the characterization of samples can hardly be overestimated. They include, amongst others, gelfiltration, analytical ultracentrifugation, static and dynamic light scattering, NMR, etc. They are quite simply indispensable for doing good and accurate science with SAS, in particular in more complex systems (macromolecular complexes, membrane proteins …). If they are not presentedin more detail in this thesis, it is not out of ignorance of this fact but due to the lack of space

Interpretation of in-solution small-angle scattering data using molecular dynamics simulations

The accurate determination of macromolecular structures often necessitates joint experimental and computational efforts. In this thesis, MD simulations are engaged to interpret small-angle scattering data of X-rays (SAXS) and neutrons (SANS). SAXS and SANS experiments are performed under near-native conditions, but provide only limited amount of structural information that are, in addition, difficult to interpret. Therefore, MD simulations are highly compatible with small-angle scattering experiments - simulations are used to interpret experimental data and in turn, experimental data are used to validate, and if necessary to guide simulations. Here, four different yet related questions are addressed. First, we quantify the influence of the ion cloud on interpreting SAXS data of charged proteins. Secondly, we study the size and the shape of detergent micelles, as this represents a starting point in improving the stability of protein-detergent complexes during the solubilization of membrane proteins. In the next step, we derive an ensemble of detergent micelles in agreement with experimental data, enabling us to study the in uence of various effects of SAXS curves and thereby making an important step towards the better understanding of the SAXS experimental data. Finally, we demonstrate how SAXS and SANS data can jointly be combined with MD simulations, allowing for fine structural characterization of proteindetergent complexes.Die akkurate Bestimmung makromolekularer Strukturen erfordert häufig experimentelle Daten mit rechnerischen Methoden zu kombinieren. In dieser Arbeit werden MD Simulationen zur Interpretation von experimentellen Daten, die mittels Kleinwinkel- Röntgenstreuung (SAXS) und Kleinwinkel-Neutronenstreuung (SANS) aufgenommen wurden, genutzt. SAXS- und SANS-Experimente werden unter nahezu natürlichen Bedingungen durchgeführt, liefern jedoch nur ein beschränktes Maß an struktureller Information, welche sich zudem auch nur schwer interpretieren lässt. MD Simulationen eignen sich besonders gut zur Kombination mit Kleinwinkelstreuexperimenten, da die Simulationen verwendet werden können, um experimentelle Daten zu interpretieren. Umgekehrt werden experimentelle Daten benutzt, um Simulationen zu validieren und sie nötigenfalls zu lenken. Im Folgenden werden vier verschiedene jedoch verbundene Fragestellungen behandelt. Im ersten Abschnitt wird der Ein uss einer lonenwolke auf die Interpretation von SAXS-Daten geladener Proteine untersucht. Danach wird die Form und Größe tensidbasierter Mizellen untersucht. Dies ist ein Ausgangspunkt, um die Stabilität von Protein-Detergenz Komplexen während des Lösens von Membranproteinen zu erhöhen. Im nächsten Schritt wird ein Ensemble tensidbasierter Mizellen bestimmt, welches mit expirmentellen Daten übereinstimmt und es ermöglicht, den Einfluss verschiedener Effekte auf die SAXS Kurve zu studieren. Dies stellt einen wichtigen Schritt dar, um experimentelle SAXS Daten besser zu verstehen. Als vierten Punkt demonstrieren wir, wie die Kombination von SAXS- und SANS-Daten zusammen mit MD-Simulation eine genaue Strukturbestimmung von Protein-Detergenz-Komplexen ermöglicht

United we stand: combining structural methods

Structural biologists benefit enormously by combining structural approaches to tackle biological systems. This is evident in the increasing use of complementary methods combined with the traditional structural biology techniques of macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) and electron microscopy (EM) to generate structural information

The Structure and Function of Photosystem I and Photosystem I – Hydrogenase Protein Fusions: An Experimental and Computational Study

Photosystem I (PSI) is a membrane protein involved in the photosynthetic cycle of plants, algae, and cyanobacteria that is of specific interest due to its ability to harness solar energy to generate reducing power. This work seeks to form an in vitro hybrid protein fusion between the membrane integral PSI protein and the membrane-bound hydrogenase (MBH) enzyme, in an effort to improve electron transport between these two proteins. Small-angle neutron scattering (SANS) was used to characterize the detergent-solubilized solution structure of trimeric PSI from the cyanobacterium Thermosynechococcus elongatus, which showed that the detergent interacts primarily with the hydrophobic periphery of PSI. The SANS results were used as a guide to constructing a model of trimeric PSI embedded in a detergent belt. Subsequent all-atom molecular dynamics (MD) simulations of the PSI-detergent complex suggested that the detergent environment could negatively impact the long-term stability of PSI, but is not likely to affect PSI activity or hinder its ligation to the MBH. Having verified that the solution structure of the PSI-detergent complex will not affect formation of PSI-MBH fusions, the membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha was genetically engineered to express a Gly3 [Gly-Gly-Gly] tag on the N-terminus of the small subunit to allow for site-specific ligation to the psaE subunit of PSI. H2 [hydrogen] uptake activity results show a complete loss of activity in the mutant R. eutropha strain, possibly due to mutations introduced during previous genetic engineering work. In parallel, MD simulations of the PSI-MBH fusion protein indicate this ligation strategy is not optimal for electron transport between these proteins. This MD approach can be used to evaluate other PSI-MBH fusion strategies, possibly targeting other stromal subunits of PSI. Finally, MD simulations of previously studied PSI-[FeFe]-hydrogenase fusions were conducted, revealing significant distortion of the protein structure that could limit their long-term stability

Insight into the Structure and Dynamics of Polymers by Neutron Scattering Combined with Atomistic Molecular Dynamics Simulations

Combining neutron scattering and fully atomistic molecular dynamics simulations allows unraveling structural and dynamical features of polymer melts at different length scales, mainly in the intermolecular and monomeric range. Here we present the methodology developed by us and the results of its application during the last years in a variety of polymers. This methodology is based on two pillars: (i) both techniques cover approximately the same length and time scales and (ii) the classical van Hove formalism allows easily calculating the magnitudes measured by neutron scattering from the simulated atomic trajectories. By direct comparison with experimental results, the simulated cell is validated. Thereafter, the information of the simulations can be exploited, calculating magnitudes that are experimentally inaccessible or extending the parameters range beyond the experimental capabilities. We show how detailed microscopic insight on structural features and dynamical processes of various kinds has been gained in polymeric systems with different degrees of complexity, and how intriguing questions as the collective behavior at intermediate length scales have been faced.This research was funded by the Basque Government, code: IT-1175-19 and the Ministerio de Economía y Competitividad code: PGC2018-094548-B-I00 (MCIU/AEI/FEDER, UE)

Outcome of the First wwPDB Hybrid / Integrative Methods Task Force Workshop

Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models