Publications by authors named "Clement E Blanchet"

18 Publications

  • Page 1 of 1

Anomalous SAXS at P12 beamline EMBL Hamburg: instrumentation and applications.

J Synchrotron Radiat 2021 May 14;28(Pt 3):812-823. Epub 2021 Apr 14.

European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.

Small-angle X-ray scattering (SAXS) is an established method for studying nanostructured systems and in particular biological macromolecules in solution. To obtain element-specific information about the sample, anomalous SAXS (ASAXS) exploits changes of the scattering properties of selected atoms when the energy of the incident X-rays is close to the binding energy of their electrons. While ASAXS is widely applied to condensed matter and inorganic systems, its use for biological macromolecules is challenging because of the weak anomalous effect. Biological objects are often only available in small quantities and are prone to radiation damage, which makes biological ASAXS measurements very challenging. The BioSAXS beamline P12 operated by the European Molecular Biology Laboratory (EMBL) at the PETRA III storage ring (DESY, Hamburg) is dedicated to studies of weakly scattering objects. Here, recent developments at P12 allowing for ASAXS measurements are presented. The beamline control, data acquisition and data reduction pipeline of the beamline were adapted to conduct ASAXS experiments. Modelling tools were developed to compute ASAXS patterns from atomic models, which can be used to analyze the data and to help designing appropriate data collection strategies. These developments are illustrated with ASAXS experiments on different model systems performed at the P12 beamline.
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http://dx.doi.org/10.1107/S1600577521003404DOI Listing
May 2021

Rapid screening of grown protein crystals via a small-angle X-ray scattering/X-ray powder diffraction synergistic approach.

J Appl Crystallogr 2020 Oct 25;53(Pt 5):1169-1180. Epub 2020 Sep 25.

Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, Lübeck 23562, Germany.

Crystallization of recombinant proteins in living cells is an exciting new approach for structural biology that provides an alternative to the time-consuming optimization of protein purification and extensive crystal screening steps. Exploiting the potential of this approach requires a more detailed understanding of the cellular processes involved and versatile screening strategies for crystals in a cell culture. Particularly if the target protein forms crystalline structures of unknown morphology only in a small fraction of cells, their detection by applying standard visualization techniques can be time consuming and difficult owing to the environmental challenges imposed by the living cells. In this study, a high-brilliance and low-background bioSAXS beamline is employed for rapid and sensitive detection of protein microcrystals grown within insect cells. On the basis of the presence of Bragg peaks in the recorded small-angle X-ray scattering profiles, it is possible to assess within seconds whether a cell culture contains microcrystals, even in a small percentage of cells. Since such information cannot be obtained by other established detection methods in this time frame, this screening approach has the potential to overcome one of the bottlenecks of intracellular crystal detection. Moreover, the association of the Bragg peak positions in the scattering curves with the unit-cell composition of the protein crystals raises the possibility of investigating the impact of environmental conditions on the crystal structure of the intracellular protein crystals. This information provides valuable insights helping to further understand the crystallization process.
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http://dx.doi.org/10.1107/S1600576720010687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534541PMC
October 2020

Investigation of pH-Responsiveness inside Lipid Nanoparticles for Parenteral mRNA Application Using Small-Angle X-ray Scattering.

Langmuir 2020 11 27;36(44):13331-13341. Epub 2020 Oct 27.

Department of Pharmaceutics and Biopharmaceutics, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Staudingerweg 5, D-55099 Mainz, Germany.

Messenger ribonucleic acid (mRNA)-based nanomedicines have shown to be a promising new lead in a broad field of potential applications such as tumor immunotherapy. Of these nanomedicines, lipid-based mRNA nanoparticles comprising ionizable lipids are gaining increasing attention as versatile technologies for fine-tuning toward a given application, with proven potential for successful development up to clinical practice. Still, several hurdles have to be overcome to obtain a drug product that shows adequate mRNA delivery and clinical efficacy. In this study, pH-induced changes in internal molecular organization and overall physicochemical characteristics of lipoplexes comprising ionizable lipids were investigated using small-angle X-ray scattering and supplementary techniques. These changes were determined for different types of ionizable lipids, present at various molar fractions and N/P ratios inside the phospholipid membranes. The investigated systems showed a lamellar organization, allowing an accurate determination of pH-dependent structural changes. The differences in the pH responsiveness of the systems comprising different ionizable lipids and mRNA fractions could be clearly revealed from their structural evolution. Measurements of the degree of ionization and pH-dependent mRNA loading into the systems by fluorescence assays supported the findings from the structural investigation. Our approach allows for direct in situ determination of the structural response of the lipoplex systems to changes of the environmental pH similar to that observed for endosomal uptake. These data therefore provide valuable complementary information for understanding and fine-tuning of tailored mRNA delivery systems toward improved cellular uptake and endosomal processing.
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http://dx.doi.org/10.1021/acs.langmuir.0c02446DOI Listing
November 2020

Hybrid Biopolymer and Lipid Nanoparticles with Improved Transfection Efficacy for mRNA.

Cells 2020 09 5;9(9). Epub 2020 Sep 5.

Department of Pharmaceutics and Biopharmaceutics, Johannes Gutenberg University Mainz, D-55131 Mainz, Germany.

Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation.
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http://dx.doi.org/10.3390/cells9092034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563888PMC
September 2020

Structural Kinetics of MsbA Investigated by Stopped-Flow Time-Resolved Small-Angle X-Ray Scattering.

Structure 2020 03 30;28(3):348-354.e3. Epub 2019 Dec 30.

The Hamburg Centre for Ultrafast Imaging, Luruper Chaussee 149, 22761 Hamburg, Germany; Department of Chemistry, Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany. Electronic address:

Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs. Our data allowed us to structurally characterize the major states involved in the process and determine time constants for NBD dimerization and dissociation. In the full-length protein, these structural transitions occur on much faster time scales, indicating close-proximity effects and structural coupling of the transmembrane domains with the NBDs.
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http://dx.doi.org/10.1016/j.str.2019.12.001DOI Listing
March 2020

Smaller capillaries improve the small-angle X-ray scattering signal and sample consumption for biomacromolecular solutions.

J Synchrotron Radiat 2018 Jul 26;25(Pt 4):1113-1122. Epub 2018 Jun 26.

European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.

Radiation damage by intense X-ray beams at modern synchrotron facilities is one of the major complications for biological small-angle X-ray scattering (SAXS) investigations of macromolecules in solution. To limit the damage, samples are typically measured under a laminar flow through a cell (typically a capillary) such that fresh solution is continuously exposed to the beam during measurement. The diameter of the capillary that optimizes the scattering-to-absorption ratio at a given X-ray wavelength can be calculated a priori based on fundamental physical properties. However, these well established scattering and absorption principles do not take into account the radiation susceptibility of the sample or the often very limited amounts of precious biological material available for an experiment. Here it is shown that, for biological solution SAXS, capillaries with smaller diameters than those calculated from simple scattering/absorption criteria allow for a better utilization of the available volumes of radiation-sensitive samples. This is demonstrated by comparing two capillary diameters d (d = 1.7 mm, close to optimal for 10 keV; and d = 0.9 mm, which is nominally sub-optimal) applied to study different protein solutions at various flow rates. The use of the smaller capillaries ultimately allows one to collect higher-quality SAXS data from the limited amounts of purified biological macromolecules.
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http://dx.doi.org/10.1107/S1600577518007907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038601PMC
July 2018

Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments.

Nat Protoc 2016 Nov 6;11(11):2122-2153. Epub 2016 Oct 6.

European Molecular Biology Laboratory (EMBL) Hamburg Outstation, DESY, Hamburg, Germany.

Small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) are techniques used to extract structural parameters and determine the overall structures and shapes of biological macromolecules, complexes and assemblies in solution. The scattering intensities measured from a sample contain contributions from all atoms within the illuminated sample volume, including the solvent and buffer components, as well as the macromolecules of interest. To obtain structural information, it is essential to prepare an exactly matched solvent blank so that background scattering contributions can be accurately subtracted from the sample scattering to obtain the net scattering from the macromolecules in the sample. In addition, sample heterogeneity caused by contaminants, aggregates, mismatched solvents, radiation damage or other factors can severely influence and complicate data analysis, so it is essential that the samples be pure and monodisperse for the duration of the experiment. This protocol outlines the basic physics of SAXS and SANS, and it reveals how the underlying conceptual principles of the techniques ultimately 'translate' into practical laboratory guidance for the production of samples of sufficiently high quality for scattering experiments. The procedure describes how to prepare and characterize protein and nucleic acid samples for both SAXS and SANS using gel electrophoresis, size-exclusion chromatography (SEC) and light scattering. Also included are procedures that are specific to X-rays (in-line SEC-SAXS) and neutrons, specifically preparing samples for contrast matching or variation experiments and deuterium labeling of proteins.
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http://dx.doi.org/10.1038/nprot.2016.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402874PMC
November 2016

LabDisk for SAXS: a centrifugal microfluidic sample preparation platform for small-angle X-ray scattering.

Lab Chip 2016 Apr;16(7):1161-70

Hahn-Schickard - Georges-Koehler-Allee 103, 79110 Freiburg, Germany.

We present a centrifugal microfluidic LabDisk for protein structure analysis via small-angle X-ray scattering (SAXS) on synchrotron beamlines. One LabDisk prepares 120 different measurement conditions, grouped into six dilution matrices. Each dilution matrix: (1) features automatic generation of 20 different measurement conditions from three input liquids and (2) requires only 2.5 μl of protein solution, which corresponds to a tenfold reduction in sample volume in comparison to the state of the art. Total hands on time for preparation of 120 different measurement conditions is less than 5 min. Read-out is performed on disk within the synchrotron beamline P12 at EMBL Hamburg (PETRA III, DESY). We demonstrate: (1) aliquoting of 40 nl aliquots for five different liquids typically used in SAXS and (2) confirm fluidic performance of aliquoting, merging, mixing and read-out from SAXS experiments (2.7-4.4% CV of protein concentration). We apply the LabDisk for SAXS for basic analysis methods, such as measurement of the radius of gyration, and advanced analysis methods, such as the ab initio calculation of 3D models. The suitability of the LabDisk for SAXS for protein structure analysis under different environmental conditions is demonstrated for glucose isomerase under varying protein and NaCl concentrations. We show that the apparent radius of gyration of the negatively charged glucose isomerase decreases with increasing protein concentration at low salt concentration. At high salt concentration the radius of gyration (Rg) does not change with protein concentrations. Such experiments can be performed by a non-expert, since the LabDisk for SAXS does not require attachment of tubings or pumps and can be filled with regular pipettes. The new platform has the potential to introduce routine high-throughput SAXS screening of protein structures with minimal input volumes to the regular operation of synchrotron beamlines.
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http://dx.doi.org/10.1039/c5lc01580dDOI Listing
April 2016

Automated pipeline for purification, biophysical and x-ray analysis of biomacromolecular solutions.

Sci Rep 2015 Jun 1;5:10734. Epub 2015 Jun 1.

European Molecular Biology Laboratory (EMBL) Hamburg, 22607 Hamburg, Germany.

Small angle X-ray scattering (SAXS), an increasingly popular method for structural analysis of biological macromolecules in solution, is often hampered by inherent sample polydispersity. We developed an all-in-one system combining in-line sample component separation with parallel biophysical and SAXS characterization of the separated components. The system coupled to an automated data analysis pipeline provides a novel tool to study difficult samples at the P12 synchrotron beamline (PETRA-3, EMBL/DESY, Hamburg).
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http://dx.doi.org/10.1038/srep10734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377070PMC
June 2015

Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY).

J Appl Crystallogr 2015 Apr 12;48(Pt 2):431-443. Epub 2015 Mar 12.

European Molecular Biology Laboratory, Hamburg Outstation , Notkestrasse 85, Hamburg, 22603, Germany.

A high-brilliance synchrotron P12 beamline of the EMBL located at the PETRA III storage ring (DESY, Hamburg) is dedicated to biological small-angle X-ray scattering (SAXS) and has been designed and optimized for scattering experiments on macromolecular solutions. Scatterless slits reduce the parasitic scattering, a custom-designed miniature active beamstop ensures accurate data normalization and the photon-counting PILATUS 2M detector enables the background-free detection of weak scattering signals. The high flux and small beam size allow for rapid experiments with exposure time down to 30-50 ms covering the resolution range from about 300 to 0.5 nm. P12 possesses a versatile and flexible sample environment system that caters for the diverse experimental needs required to study macromolecular solutions. These include an in-vacuum capillary mode for standard batch sample analyses with robotic sample delivery and for continuous-flow in-line sample purification and characterization, as well as an in-air capillary time-resolved stopped-flow setup. A novel microfluidic centrifugal mixing device (SAXS disc) is developed for a high-throughput screening mode using sub-microlitre sample volumes. Automation is a key feature of P12; it is controlled by a beamline meta server, which coordinates and schedules experiments from either standard or nonstandard operational setups. The integrated SASFLOW pipeline automatically checks for consistency, and processes and analyses the data, providing near real-time assessments of overall parameters and the generation of low-resolution models within minutes of data collection. These advances, combined with a remote access option, allow for rapid high-throughput analysis, as well as time-resolved and screening experiments for novice and expert biological SAXS users.
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http://dx.doi.org/10.1107/S160057671500254XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379436PMC
April 2015

A small and robust active beamstop for scattering experiments on high-brilliance undulator beamlines.

J Synchrotron Radiat 2015 Mar 4;22(2):461-4. Epub 2015 Feb 4.

European Molecular Biology Laboratory, Hamburg Outstation c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany.

A small active in-vacuum beamstop has been developed to monitor the flux of intense third-generation synchrotron X-ray beams protecting the downstream detector from the direct beam. Standard active beamstops, where a built-in diode directly absorbs the beam, have limitations in size and lifetime. In the present design, a silicon PIN diode detects the photons back-scattered from a cavity in the beamstop. This approach drastically reduces the radiation dose on the diode and thus increases its lifetime. The beamstop with a diameter of 2 mm has been fabricated to meet the requirements for the P12 bioSAXS beamline of EMBL Hamburg at PETRA III (DESY). The beamstop is in regular user operation at the beamline and displays a good response over the range of energies tested (6-20 keV). Further miniaturization of the diode is easily possible as its size is not limited by the PIN diode used.
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http://dx.doi.org/10.1107/S160057751402829XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344362PMC
March 2015

Limiting radiation damage for high-brilliance biological solution scattering: practical experience at the EMBL P12 beamline PETRAIII.

J Synchrotron Radiat 2015 Mar 4;22(2):273-9. Epub 2015 Feb 4.

European Molecular Biology Laboratory Hamburg Outstation, c/o Deutsches Elektronen-Synchrotron, Notkestrasse 85, Hamburg 22603, Germany.

Radiation damage is the general curse of structural biologists who use synchrotron small-angle X-ray scattering (SAXS) to investigate biological macromolecules in solution. The EMBL-P12 biological SAXS beamline located at the PETRAIII storage ring (DESY, Hamburg, Germany) caters to an extensive user community who integrate SAXS into their diverse structural biology programs. The high brilliance of the beamline [5.1 × 10(12) photons s(-1), 10 keV, 500 (H) µm × 250 (V) µm beam size at the sample position], combined with automated sample handling and data acquisition protocols, enable the high-throughput structural characterization of macromolecules in solution. However, considering the often-significant resources users invest to prepare samples, it is crucial that simple and effective protocols are in place to limit the effects of radiation damage once it has been detected. Here various practical approaches are evaluated that users can implement to limit radiation damage at the P12 beamline to maximize the chances of collecting quality data from radiation sensitive samples.
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http://dx.doi.org/10.1107/S1600577515000375DOI Listing
March 2015

BioSAXS Sample Changer: a robotic sample changer for rapid and reliable high-throughput X-ray solution scattering experiments.

Acta Crystallogr D Biol Crystallogr 2015 Jan 1;71(Pt 1):67-75. Epub 2015 Jan 1.

European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble, France.

Small-angle X-ray scattering (SAXS) of macromolecules in solution is in increasing demand by an ever more diverse research community, both academic and industrial. To better serve user needs, and to allow automated and high-throughput operation, a sample changer (BioSAXS Sample Changer) that is able to perform unattended measurements of up to several hundred samples per day has been developed. The Sample Changer is able to handle and expose sample volumes of down to 5 µl with a measurement/cleaning cycle of under 1 min. The samples are stored in standard 96-well plates and the data are collected in a vacuum-mounted capillary with automated positioning of the solution in the X-ray beam. Fast and efficient capillary cleaning avoids cross-contamination and ensures reproducibility of the measurements. Independent temperature control for the well storage and for the measurement capillary allows the samples to be kept cool while still collecting data at physiological temperatures. The Sample Changer has been installed at three major third-generation synchrotrons: on the BM29 beamline at the European Synchrotron Radiation Facility (ESRF), the P12 beamline at the PETRA-III synchrotron ([email protected]) and the I22/B21 beamlines at Diamond Light Source, with the latter being the first commercial unit supplied by Bruker ASC.
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http://dx.doi.org/10.1107/S1399004714026959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304687PMC
January 2015

Structural determinants and mechanism of mammalian CRM1 allostery.

Structure 2013 Aug 11;21(8):1350-60. Epub 2013 Jul 11.

Abteilung für Theoretische und Computergestützte Biophysik, Max-Planck-Institut für Biophysikalische Chemie, Am Faßberg 11, 37077 Göttingen, Germany.

Proteins carrying nuclear export signals cooperatively assemble with the export factor CRM1 and the effector protein RanGTP. In lower eukaryotes, this cooperativity is coupled to CRM1 conformational changes; however, it is unknown if mammalian CRM1 maintains its compact conformation or shows similar structural flexibility. Here, combinations of small-angle X-ray solution scattering and electron microscopy experiments with molecular dynamics simulations reveal pronounced conformational flexibility in mammalian CRM1 and demonstrate that RanGTP binding induces association of its N- and C-terminal regions to form a toroid structure. The CRM1 toroid is stabilized mainly by local interactions between the terminal regions, rather than by global strain. The CRM1 acidic loop is key in transmitting the effect of this RanGTP-induced global conformational change to the NES-binding cleft by shifting its population to the open state, which displays enhanced cargo affinity. Cooperative CRM1 export complex assembly thus constitutes a highly dynamic process, encompassing an intricate interplay of global and local structural changes.
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http://dx.doi.org/10.1016/j.str.2013.05.015DOI Listing
August 2013

Quaternary structure of human, Drosophila melanogaster and Caenorhabditis elegans MFE-2 in solution from synchrotron small-angle X-ray scattering.

FEBS Lett 2013 Feb 8;587(4):305-10. Epub 2013 Jan 8.

Department of Biochemistry and Biocenter Oulu, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland.

Multifunctional enzyme type 2 (MFE-2) forms part of the fatty acid β-oxidation pathway in peroxisomes. MFE-2s from various species reveal proteins with structurally homologous functional domains assembled in different compilations. Crystal structures of all domain types are known. SAXS data from human, fruit fly and Caenorhabditiselegans MFE-2s and their constituent domains were collected, and both ab initio and rigid body models constructed. Location of the putative substrate binding helper domain SCP-2L (sterol carrier protein 2-like), which is not part of MFE-2 protein in every species and not seen as part of any previous MFE-2 structures, was determined. The obtained models of human and C. elegans MFE-2 lend a direct structural support to the idea of the biological role of SCP-2L.
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http://dx.doi.org/10.1016/j.febslet.2012.12.014DOI Listing
February 2013

Small-angle X-ray scattering on biological macromolecules and nanocomposites in solution.

Annu Rev Phys Chem 2013 3;64:37-54. Epub 2012 Dec 3.

European Molecular Biology Laboratory Hamburg, 22603 Hamburg, Germany.

Small-angle X-ray scattering (SAXS) is a powerful method to study the structural properties of materials at the nanoscale. Recent progress in instrumentation and analysis methods has led to rapidly growing applications of this technique for the characterization of biological macromolecules in solution. Ab initio and rigid-body modeling methods allow one to build three-dimensional, low-resolution models from SAXS data. With the new approaches, oligomeric states of proteins and macromolecular complexes can be assessed, chemical equilibria and kinetic reactions can be studied, and even flexible objects such as intrinsically unfolded proteins can be quantitatively characterized. This review describes the analysis methods of SAXS data from macromolecular solutions, ranging from the computation of overall structural parameters to advanced three-dimensional modeling. The efficiency of these methods is illustrated by recent applications to biological macromolecules and nanocomposite particles.
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http://dx.doi.org/10.1146/annurev-physchem-040412-110132DOI Listing
September 2013

Amyloid fibrils formed by the programmed cell death regulator Bcl-xL.

J Mol Biol 2012 Jan 19;415(3):584-99. Epub 2011 Nov 19.

CEA, DSV, iRTSV, Laboratoire de Chimie et Biologie des Métaux (UMR 5249), CEA Grenoble, 17 Rue des Martyrs, Grenoble, F-38054, France.

The accumulation of amyloid fibers due to protein misfolding is associated with numerous human diseases. For example, the formation of amyloid deposits in neurodegenerative pathologies is correlated with abnormal apoptosis. We report here the in vitro formation of various types of aggregates by Bcl-xL, a protein of the Bcl-2 family involved in the regulation of apoptosis. Bcl-xL forms aggregates in three states, micelles, native-like fibrils, and amyloid fibers, and their biophysical characterization has been performed in detail. Bcl-xL remains in its native state within micelles and native-like fibrils, and our results suggest that native-like fibrils are formed by the association of micelles. Formation of amyloid structures, that is, nonnative intermolecular β-sheets, is favored by the proximity of proteins within fibrils at the expense of the Bcl-xL native structure. Finally, we provide evidence of a direct relationship between the amyloid character of the fibers and the tertiary-structure stability of the native Bcl-xL. The potential causality between the accumulation of Bcl-xL into amyloid deposits and abnormal apoptosis during neurodegenerative diseases is discussed.
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http://dx.doi.org/10.1016/j.jmb.2011.11.024DOI Listing
January 2012

Cofactor effects on the protein folding reaction: acceleration of alpha-lactalbumin refolding by metal ions.

Protein Sci 2006 Apr 7;15(4):659-71. Epub 2006 Mar 7.

Laboratoire de Biophysique Moléculaire et Cellulaire, Unité Mixte de Recherche 5090, Département Réponse et Dynamique Cellulaires, CEA-Grenoble, 38054 Grenoble cedex 9, France.

About 30% of proteins require cofactors for their proper folding. The effects of cofactors on the folding reaction have been investigated with alpha-lactalbumin as a model protein and metal ions as cofactors. Metal ions accelerate the refolding of alpha-lactalbumin by lessening the energy barrier between the molten globule state and the transition state, mainly by decreasing the difference of entropy between the two states. These effects are linked to metal ion binding to the protein in the native state. Hence, relationships between the metal affinities for the intermediate states and those for the native state are observed. Some residual specificity for the calcium ion is still observed in the molten globule state, this specificity getting closer in the transition state to that of the native state. The comparison between kinetic and steady-state data in association with the Phi value method indicates the binding of the metal ions on the unfolded state of alpha-lactalbumin. Altogether, these results provide insight into cofactor effects on protein folding. They also suggest new possibilities to investigate the presence of residual native structures in the unfolded state of protein and the effects of such structures on the protein folding reaction and on protein stability.
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http://dx.doi.org/10.1110/ps.051904206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242491PMC
April 2006