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

Structure of the K-turn U4 RNA: a combined NMR and SANS study

K-turn motifs are universal RNA structural elements providing a binding platform for proteins in several cellular contexts. Their characteristic is a sharp kink in the phosphate backbone that puts the two helical stems of the protein-bound RNA at an angle of 60°. However, to date no high-resolution structure of a naked K-turn motif is available. Here, we present the first structural investigation at atomic resolution of an unbound K-turn RNA (the spliceosomal U4-Kt RNA) by a combination of NMR and small-angle neutron scattering data. With this study, we wish to address the question whether the K-turn structural motif assumes the sharply kinked conformation in the absence of protein binders and divalent cations. Previous studies have addressed this question by fluorescence resonance energy transfer, biochemical assays and molecular dynamics simulations, suggesting that the K-turn RNAs exist in equilibrium between a kinked conformation, which is competent for protein binding, and a more extended conformation, with the population distribution depending on the concentration of divalent cations. Our data shows that the U4-Kt RNA predominantly assumes the more extended conformation in the absence of proteins and divalent cations. The internal loop region is well structured but adopts a different conformation from the one observed in complex with proteins. Our data suggests that the K-turn consensus sequence does not per se code for the kinked conformation; instead the sharp backbone kink requires to be stabilized by protein binders

A real-time analysis of GFP unfolding by the AAA+ unfoldase PAN

Protein quality control systems are essential to maintain a healthy proteome. They often consist of an unfoldase unit, typically an AAA+ ATPase, coupled with a protease unit. In all kingdoms of life, they function to eliminate misfolded proteins, and thus prevent that their aggregates do harm to the cell, and to rapidly regulate protein levels in the presence of environmental changes. Despite the huge progress made in the past two decades in understanding the mechanism of function of protein degradation systems, the fate of the substrate during the unfolding and proteolytic processes remains poorly understood. Here we exploit an NMR-based approach to monitor GFP processing by the archaeal PAN unfoldase and the PAN–20S degradation system in real time. We find that PAN-dependent unfolding of GFP does not involve the release of partially-folded GFP molecules resulting from futile unfolding attempts. In contrast, once stably engaged with PAN, GFP molecules are efficiently transferred to the proteolytic chamber of the 20S subunit, despite the only weak affinity of PAN for the 20S subunit in the absence of substrate. This is essential to guarantee that unfolded but not proteolyzed proteins are not released into solution, where they would form toxic aggregates. The results of our studies are in good agreement with previous results derived from real-time small-angle-neutron-scattering experiments and have the advantage of allowing the investigation of substrates and products at amino-acid resolution

An NMR Study of a 300-kDa AAA+ Unfoldase

AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C2 symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C2 symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution

Conformational States of a Bacterial α2-Macroglobulin Resemble Those of Human Complement C3

α2 macroglobulins (α2Ms) are broad-spectrum protease inhibitors that play essential roles in the innate immune system of eukaryotic species. These large, multi-domain proteins are characterized by a broad-spectrum bait region and an internal thioester, which, upon cleavage, becomes covalently associated to the target protease, allowing its entrapment by a large conformational modification. Notably, α2Ms are part of a larger protein superfamily that includes proteins of the complement system, such as C3, a multi-domain macromolecule which is also characterized by an internal thioester-carrying domain and whose activation represents the pivotal step in the complement cascade. Recently, α2M/C3-like genes were identified in a large number of bacterial genomes, and the Escherichia coli α2M homolog (ECAM) was shown to be activated by proteases. In this work, we have structurally characterized ECAM by electron microscopy and small angle scattering (SAXS) techniques. ECAM is an elongated, flexible molecule with overall similarities to C3 in its inactive form; activation by methylamine, chymotrypsin, or elastase induces a conformational modification reminiscent of the one undergone by the transformation of C3 into its active form, C3b. In addition, the proposed C-terminus of ECAM displays high flexibility and different conformations, and could be the recognition site for partner macromolecules. This work sheds light on a potential bacterial defense mechanism that mimics structural rearrangements essential for activation of the complement cascade in eukaryotes, and represents a possible novel target for the development of antibacterials

Structure and interactions of fish type III antifreeze protein in solution

It has been suggested that above a critical protein concentration, fish Type III antifreeze protein (AFP III) selfassembles to form micelle-like structures that may play a key role In antifreeze activity. To understand the complex activity of AFP III, a comprehensive description of its association state and structural organization in solution is necessary. We used analytical ultracentrifugation, analytical size-exclusion chromatography, and dynamic light scattering to characterize the interactions and homogeneity of AFP III in solution. Small-angle neutron scattering was used to determine the low-resolution structure in solution. Our results clearly show that at concentrations up to 20 mg mL-1 and at temperatures of 20°C, 6°C, and 4°C, AFP III is monomeric in solution and adopts a structure compatible with that determined by crystallography. Surface tension measurements show a propensity of AFP III to localize at the air/water interface, but this surface activity is not correlated with any aggregation in the bulk. These results support the hypothesis that each AFP III molecule acts independently of the others, and that specific intermolecular interactions between monomers are not required for binding to ice. The lack of attractive interactions between monomers may be functionally important, allowing for more efficient binding and covering of the ice surface.Instituto de Física de Líquidos y Sistemas Biológico

Assessing the conformational changes of pb5, the receptor-binding protein of phage T5, upon binding to its Escherichia coli receptor FhuA

Within tailed bacteriophages, interaction of the receptor-binding protein (RBP) with the target cell triggers viral DNA ejection into the host cytoplasm. In the case of phage T5, the RBP pb5 and the receptor FhuA, an outer membrane protein of Escherichia coli, have been identified. Here, we use small angle neutron scattering and electron microscopy to investigate the FhuA-pb5 complex. Specific deuteration of one of the partners allows the complete masking in small angle neutron scattering of the surfactant and unlabeled proteins when the complex is solubilized in the fluorinated surfactant F6-DigluM. Thus, individual structures within a membrane protein complex can be described. The solution structure of FhuA agrees with its crystal structure; that of pb5 shows an elongated shape. Neither displays significant conformational changes upon interaction. The mechanism of signal transduction within phage T5 thus appears different from that of phages binding cell wall saccharides, for which structural information is available

Gate-tunable, normally-on to normally-off memristance transition inpatterned LaAlO3/SrTiO3 interfaces

The authors gratefully acknowledge the support from the state of Bavaria as well as from the Deutsche Forschungsgemeinschaft (FOR1162 and SFB1170).We report gate-tunable memristive switching in patterned LaAlO3/SrTiO3 interfaces at cryogenic temperatures. The application of voltages in the order of a few volts to the back gate of the device allows controlling and switching on and -off the inherent memory functionality (memristance). For large and small gate voltages a simple non-linear resistance characteristic is observed while a pinched hysteresis loop and memristive switching occurs in an intermediate voltage range. The memristance is further controlled by the density of oxygen vacancies, which is tuned by annealing the sample at 300 °C in nitrogen atmosphere. Depending on the annealing time the memristance at zero gate voltage can be switched on and off leading to normally-on and normally-off memristors. The presented device offers reversible and irreversible control of memristive characteristics by gate voltages and annealing, respectively, which may allow to compensate fabrication variabilities of memristors that complicate the realization of large memristor-based neural networks.PostprintPeer reviewe

Structure and interactions of fish type III antifreeze protein in solution

It has been suggested that above a critical protein concentration, fish Type III antifreeze protein (AFP III) selfassembles to form micelle-like structures that may play a key role In antifreeze activity. To understand the complex activity of AFP III, a comprehensive description of its association state and structural organization in solution is necessary. We used analytical ultracentrifugation, analytical size-exclusion chromatography, and dynamic light scattering to characterize the interactions and homogeneity of AFP III in solution. Small-angle neutron scattering was used to determine the low-resolution structure in solution. Our results clearly show that at concentrations up to 20 mg mL-1 and at temperatures of 20°C, 6°C, and 4°C, AFP III is monomeric in solution and adopts a structure compatible with that determined by crystallography. Surface tension measurements show a propensity of AFP III to localize at the air/water interface, but this surface activity is not correlated with any aggregation in the bulk. These results support the hypothesis that each AFP III molecule acts independently of the others, and that specific intermolecular interactions between monomers are not required for binding to ice. The lack of attractive interactions between monomers may be functionally important, allowing for more efficient binding and covering of the ice surface.Instituto de Física de Líquidos y Sistemas Biológico