A hypothesis to reconcile the physical and chemical unfolding of proteins

High pressure (HP) or urea is commonly used to disturb folding species. Pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the protein–solvent system. However, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein–urea interactions are considered to be crucial. Here, we provide NMR spectroscopy and 3D reconstructions from X-ray scattering to develop the “push-and-pull” hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by HP. In studying MpNep2 from Moniliophthora perniciosa, we tracked two cooperative units using HP-NMR as MpNep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. At subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by NMR intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle X-ray scattering (SAXS)]. This state has a higher susceptibility to pressure denaturation (lower p1/2 and larger ΔVu); thus, urea and HP share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. These observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes.

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Combining X-ray scattering and spectroscopy as structure resolving tool

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314089256 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1074-C1074

Author(s):

Sylvio Haas ◽

Thomás Plivelic ◽

Cedric Dicko

Keyword(s):

Molecular Mechanisms ◽

Basic Research ◽

Sample Volume ◽

Complex Behaviour ◽

X Ray ◽

Complex Interactions ◽

X Ray Scattering ◽

On Line ◽

Set Up ◽

Ray Scattering

Modern applications and basic research in medicine, biotechnology and materials are often concerned with hybrid (organic-inorganic) and synergetic systems. In other words, systems that brings enhanced properties and performances. The challenge is now in the understanding of the complex interactions leading to their assembly and operation. Due to their inherent chemical and structural complexities a combination of several techniques is necessary to determine unambiguously the molecular mechanisms of assembly and operation. [1, 2] To address this need, we have implemented a simultaneous measurement platform called SURF [3] that consists of SAXS, UV-Vis, Raman and fluorescence techniques. The SURF platform provides simultaneous measurements on the same sample volume and a multivariate framework to associate the spectroscopic and X-ray scattering information. Convex constraint analysis (CCA) and two dimensional correlation analyses (2DCOS and 2DHCOS) had been introduced to enhance the interpretation and integration of the data from the different techniques producing self-consistent models and resolving complex behaviour details (structure and chemistry). Additional benefits of the SURF are sample quality control and "on line" diagnostics. In this contribution, we illustrate the benefits of the SURF approach on selected examples. Acknowledgements: The SURF set-up has been mainly financial supported by MAX IV laboratory. S. Haas has a postdoctoral grant of MAX IV lab. S Canton and Q. Zhu are acknowledged for fruitful discussions.

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HU multimerization shift controls nucleoid compaction

Science Advances ◽

10.1126/sciadv.1600650 ◽

2016 ◽

Vol 2 (7) ◽

pp. e1600650 ◽

Cited By ~ 73

Author(s):

Michal Hammel ◽

Dhar Amlanjyoti ◽

Francis E. Reyes ◽

Jian-Hua Chen ◽

Rochelle Parpana ◽

...

Keyword(s):

Crystal Structures ◽

Molecular Mechanisms ◽

Bacterial Chromosome ◽

Binding Modes ◽

Dna Complexes ◽

X Ray ◽

X Ray Scattering ◽

Nucleoid Structure ◽

Ray Scattering

Molecular mechanisms controlling functional bacterial chromosome (nucleoid) compaction and organization are surprisingly enigmatic but partly depend on conserved, histone-like proteins HUαα and HUαβ and their interactions that span the nanoscale and mesoscale from protein-DNA complexes to the bacterial chromosome and nucleoid structure. We determined the crystal structures of these chromosome-associated proteins in complex with native duplex DNA. Distinct DNA binding modes of HUαα and HUαβ elucidate fundamental features of bacterial chromosome packing that regulate gene transcription. By combining crystal structures with solution x-ray scattering results, we determined architectures of HU-DNA nucleoproteins in solution under near-physiological conditions. These macromolecular conformations and interactions result in contraction at the cellular level based on in vivo imaging of native unlabeled nucleoid by soft x-ray tomography upon HUβ and ectopic HUα38 expression. Structural characterization of charge-altered HUαα-DNA complexes reveals an HU molecular switch that is suitable for condensing nucleoid and reprogramming noninvasiveEscherichia coliinto an invasive form. Collective findings suggest that shifts between networking and cooperative and noncooperative DNA-dependent HU multimerization control DNA compaction and supercoiling independently of cellular topoisomerase activity. By integrating x-ray crystal structures, x-ray scattering, mutational tests, and x-ray imaging that span from protein-DNA complexes to the bacterial chromosome and nucleoid structure, we show that defined dynamic HU interaction networks can promote nucleoid reorganization and transcriptional regulation as efficient general microbial mechanisms to help synchronize genetic responses to cell cycle, changing environments, and pathogenesis.

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The Structural Role of Gangliosides: Insights from X-ray Scattering on Model Membranes

Current Medicinal Chemistry ◽

10.2174/0929867327666200103093340 ◽

2020 ◽

Vol 27 (38) ◽

pp. 6548-6570

Author(s):

Konstantin Andreev

Keyword(s):

Cholera Toxin ◽

Molecular Mechanisms ◽

Hexagonal Lattice ◽

Langmuir Monolayers ◽

Biologically Active ◽

Adequate Model ◽

X Ray ◽

X Ray Scattering ◽

Ray Scattering

Background: Gangliosides are an essential component of eukaryotic plasma membranes implicated in multiple physiological processes. Little is known about molecular mechanisms underlying the distribution and functions of membrane gangliosides. The overwhelmingly complex organization of glycocalyx impedes the structural analysis on cell surface and the interplay between the lipid components. Advanced X-ray analytical tools applicable to studying biological interfaces call for the simplistic models that mimic ganglioside-enriched cellular membranes. Objective: To summarize the mechanistic evidences of ganglioside interactions with lipid environment and biologically active ligands using high-resolution synchrotron X-ray scattering. Methods: A comprehensive review of studies published over the last decade was done to discuss recent accomplishments and future trends. Results: Langmuir monolayers represent an adequate model system to assess the effect of gangliosides on membrane structure. Grazing incidence X-ray diffraction reveals a condensation effect by gangliosides on zwitterionic phospholipids with the cooperative packing of sialo- and phosphate groups. In turn, the arrangement of negatively charged lipids in ganglioside mixture remains unchanged due to the stretched conformation of carbohydrate moieties. Upon interaction with biological ligands, such as cholera toxin and galectins, the ganglioside redistribution within the ordered regions of monolayer follows distinct mechanistic patterns. The cholera toxin pentamer attached to the oligosaccharide core induces local transition from oblique to the hexagonal lattice resulting in phase coexistence. The incorporation of the A subunit responsible for endocytosis is further promoted by the acidic environment characteristic for endosomal space. X-ray reflectivity shows in-plane orientation of galectin dimers with the spatial mismatch between the lectin binding sites and ganglioside carbohydrates to perturb ceramide alkyl chains. Recent data also demonstrate sialic acid groups to be potential targets for novel peptide mimicking anticancer therapeutics. Conclusion: Coupled with surface X-ray scattering, the membrane mimetic approach allows for better understanding the biological role of gangliosides and their potential applications.

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Fine structure and chemical shifts in nonresonant inelastic x-ray scattering from Li-intercalated graphite

Applied Physics Letters ◽

10.1063/1.2752755 ◽

2007 ◽

Vol 91 (3) ◽

pp. 031904 ◽

Cited By ~ 39

Author(s):

M. Balasubramanian ◽

C. S. Johnson ◽

J. O. Cross ◽

G. T. Seidler ◽

T. T. Fister ◽

...

Keyword(s):

Fine Structure ◽

Chemical Shifts ◽

X Ray ◽

Intercalated Graphite ◽

X Ray Scattering ◽

Ray Scattering

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Revealing the molecular origins of fibrin’s elastomeric properties by in situ X-ray scattering

10.1101/797464 ◽

2019 ◽

Author(s):

Bart E. Vos ◽

Cristina Martinez-Torres ◽

Federica Burla ◽

John W. Weisel ◽

Gijsje H. Koenderink

Keyword(s):

Molecular Mechanisms ◽

X Ray ◽

Packing Structure ◽

X Ray Scattering ◽

Fibrin Networks ◽

Length Increase ◽

Blood Clots ◽

Structural Responses ◽

Ray Scattering

Fibrin is an elastomeric protein forming highly extensible fiber networks that provide the scaffold of blood clots. Here we reveal the molecular mechanisms that explain the large extensibility of fibrin networks by performingin situsmall angle X-ray scattering measurements while applying a shear deformation. We simultaneously measure shear-induced alignment of the fibers and changes in their axially ordered molecular packing structure. We show that fibrin networks exhibit distinct structural responses that set in consecutively as the shear strain is increased. They exhibit an entropic response at small strains (<5%), followed by progressive fiber alignment (>25% strain) and finally changes in the fiber packing structure at high strain (>100%). Stretching reduces the fiber packing order and slightly increases the axial periodicity, indicative of molecular unfolding. However, the axial periodicity changes only by 0.7%, much less than the 80% length increase of the fibers, indicating that fiber elongation mainly stems from uncoiling of the natively disordered αC-peptide linkers that laterally bond the molecules. Upon removal of the load, the network structure returns to the original isotropic state, but the fiber structure becomes more ordered and adopts a smaller packing periodicity compared to the original state. We conclude that the hierarchical packing structure of fibrin fibers, with built-in disorder, makes the fibers extensible and allows for mechanical annealing. Our results provide a basis for interpreting the molecular basis of haemostatic and thrombotic disorders associated with clotting and provide inspiration to design resilient bio-mimicking materials.

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Sub-electron-volt chemical shifts and strong interference effects measured in the resonance x-ray scattering spectra of aniline

Physical Review A ◽

10.1103/physreva.52.3730 ◽

1995 ◽

Vol 52 (5) ◽

pp. 3730-3736 ◽

Cited By ~ 36

Author(s):

Yi Luo ◽

Hans Ågren ◽

Jinghua Guo ◽

Per Skytt ◽

Nial Wassdahl ◽

...

Keyword(s):

Chemical Shifts ◽

Interference Effects ◽

X Ray ◽

Electron Volt ◽

Strong Interference ◽

X Ray Scattering ◽

Scattering Spectra ◽

Ray Scattering

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Soft X-ray absorption spectroscopy and resonant inelastic X-ray scattering spectroscopy below 100 eV: probing first-row transition-metalM-edges in chemical complexes

Journal of Synchrotron Radiation ◽

10.1107/s0909049513003142 ◽

2013 ◽

Vol 20 (4) ◽

pp. 614-619 ◽

Cited By ~ 4

Author(s):

Hongxin Wang ◽

Anthony T. Young ◽

Jinghua Guo ◽

Stephen P. Cramer ◽

Stephan Friedrich ◽

...

Keyword(s):

Metal Complexes ◽

Absorption Spectroscopy ◽

Chemical Shifts ◽

High Sensitivity ◽

Superconducting Tunnel Junction ◽

X Ray ◽

X Ray Scattering ◽

X Ray Absorption ◽

Cu Complexes ◽

Ray Scattering

X-ray absorption and scattering spectroscopies involving the 3dtransition-metalK- andL-edges have a long history in studying inorganic and bioinorganic molecules. However, there have been very few studies using theM-edges, which are below 100 eV. Synchrotron-based X-ray sources can have higher energy resolution atM-edges.M-edge X-ray absorption spectroscopy (XAS) and resonant inelastic X-ray scattering (RIXS) could therefore provide complementary information toK- andL-edge spectroscopies. In this study,M2,3-edge XAS on several Co, Ni and Cu complexes are measured and their spectral information, such as chemical shifts and covalency effects, are analyzed and discussed. In addition,M2,3-edge RIXS on NiO, NiF2and two other covalent complexes have been performed and differentd–dtransition patterns have been observed. Although still preliminary, this work on 3dmetal complexes demonstrates the potential to useM-edge XAS and RIXS on more complicated 3dmetal complexes in the future. The potential for using high-sensitivity and high-resolution superconducting tunnel junction X-ray detectors below 100 eV is also illustrated and discussed.

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Microtubule nucleation sequence studied by time-resolved x-ray scattering and electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077402 ◽

1983 ◽

Vol 41 ◽

pp. 766-767

Author(s):

Eva-Maria Mandelkow ◽

Eckhard Mandelkow ◽

Joan Bordas

Keyword(s):

Electron Microscopy ◽

Dynamic Equilibrium ◽

Time Resolved ◽

X Ray ◽

Additional Information ◽

X Ray Scattering ◽

Low Concentrations ◽

Microtubule Growth ◽

Ray Patterns ◽

Ray Scattering

When a solution of microtubule protein is changed from non-polymerising to polymerising conditions (e.g. by temperature jump or mixing with GTP) there is a series of structural transitions preceding microtubule growth. These have been detected by time-resolved X-ray scattering using synchrotron radiation, and they may be classified into pre-nucleation and nucleation events. X-ray patterns are good indicators for the average behavior of the particles in solution, but they are difficult to interpret unless additional information on their structure is available. We therefore studied the assembly process by electron microscopy under conditions approaching those of the X-ray experiment. There are two difficulties in the EM approach: One is that the particles important for assembly are usually small and not very regular and therefore tend to be overlooked. Secondly EM specimens require low concentrations which favor disassembly of the particles one wants to observe since there is a dynamic equilibrium between polymers and subunits.

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Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181269 ◽

1990 ◽

Vol 48 (1) ◽

pp. 502-503

Author(s):

Eva-Maria Mandelkow ◽

Ron Milligan

Keyword(s):

Electron Microscopy ◽

Dynamic Instability ◽

Entire Solution ◽

Reaction Scheme ◽

Time Resolved ◽

X Ray ◽

X Ray Scattering ◽

A Chain ◽

Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

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