Small-angle X-ray and neutron scatterings (SAXS and SANS; collectively called SAS) offer overwhelming opportunities for structural analysis of a biomacromolecule in solution. Modern SAS analysis with a computational simulation provides a three-dimensional structural model, whereas it is essential for the high-quality analysis to obtain the scattering profile surely corresponding to a target molecule. However, an undesirable aggregate, even at the low weight fraction, deteriorates the scattering profile of the target molecule and then lead to failure of the structural analysis. To overcome the aggregation-problem, we have developed the integrated approach with analytical ultracentrifugation (AUC) and SAS, namely “AUC-SAS”[1].

Figure 1 demonstrates the aggregation-removal with AUC-SAS for a bovin serum albumin (BSA) solution including the aggregates. AUC revealed the weight fractions of the monomer and their aggregates in the solution (Figure 1a). Because SAS offered the scattering profile ensemble-averaged over the monomer and their aggregates (open circles in Figure 1b), the simple experimental scattering profile led to the incorrect structural model as the monomer. AUC-SAS derived the scattering profile of the monomer (closed circles in Figure 1b) from the simple experimental one utilizing the information of AUC. Consequently, the derived profile led to the reasonable structural model as the monomer. In the recent progress, AUC-SAS succeeded for the solution including aggregates up to 20 % of weight fraction.

AUC-SAS does not require a large amount of sample nor very high intensity beam compared with size exclusion chromatography-SAXS (SEC-SAXS). Therefore, AUC-SAS has a potential as a complementary method for laboratory-based SAXS and standard SANS. The software for AUC-SAS data reduction is available at http://www.rri.kyoto-u.ac.jp/NSBNG/activity.html.

Proteins are bio-macromolecules that are responsible for various biological phenomena. Their functional expression takes place in the intracellular environment, i.e., the solution environment, which is a multi-component system with multiple proteins. Solution scattering is an effective method for structural analysis in such an environment. Particularly, in order to selectively analyze the structure of a specific protein in a multi-component system, the "inverse contrast matching small-angle neutron scattering (iCM-SANS)" method is valuable utilizing the characteristics of neutron scattering, in which the scattering lengths of hydrogen and deuterium differ greatly. In this method, when deuterated and hydrogenated proteins are observed in D₂O solvent, the deuterated proteins become scatteringly invisible due to contrast matching, and only the scattering of the hydrogenated proteins be observed. For this analysis, it is necessary to prepare the proteins whose the degree of deuteration is precisely controlled to 75%.

We have prepared 75% deuterated proteins using E. coli expression system and established a simple and rapid method to measure the degree of deuteration of proteins by mass spectrometry using MALDI-TOF MS. We also established a precise and simple method to measure the D₂O/H₂O ratio of the solvent using the measurement by Fourier transform infrared spectroscopy (FT-IR).

By using these techniques, we were able to obtain the accurate degree of deuteration of the proteins and D₂O/H₂O ratio in the solvent, and match out the deuterated protein very well.

Both the preservation of transparency and high refractive index is indispensable for the maintenance of normal function of eye lens. Especially, its high refractive index is attained by high protein concentration (~300 mg/mL in human eye lens). Since there exists no turnover in the eye lens, the eye lens is always at the risk of onset of aggregation. However, the long-term transparency in eye lens is preserved at least for several tens years. Then, how can eye lens maintain a long-term transparency? Key protein retarding the onset of abnormal aggregation is chaperone activity of alpha-crystallin, which exists as oligomers consisting of approximately 20~40 subunits of two homologues: alphaA-crystallin and alphaB-crystallin. Aiming at the elucidation of mechanism of its chaperone function, clarification of its quaternary structure has been challenged through crystallography techniques for long time. However, its quaternary structure has not been solved due to the availability of its crystal. To overcome such a situation, experimental trials revealing its quaternary structure have been tackled through state-of-the art experimental techniques. However, no consensus conclusions on such revealed structure have been drawn at present. We then reached one assumption that alpha-crsytallin intrinsically lacks robust quaternary structure (dynamic quaternary structure) to understand such diverse experimental results without inconsistency. It is also considered that such dynamic quaternary structure must be originated from subunit exchange between alpha-crsytallin oligomers.
To prove our expectation, we then try to apply deuteration assisted small-angle neutron scattering technique for visualizing subunit exchange in alpha-crsytallin oligomer. At the presentation, we will also discuss the effect of concentration on mechanism of subunit exchange in alpha-crsytallin oligomer.

The small angle X-ray scattering (SAXS) profile of a biomolecule reflects its meso- and nano- scale structure. Since the profile is contributed by all molecules in solution, the SAXS is a powerful method to study structural ensemble of the protein. We are establishing methods to elucidate structural ensemble of proteins at near-atomic resolution by combining SAXS and molecular dynamics simulations.

In this study we focused on structure and dynamics of multi-domain protein ER-60. ER-60 is a member of Protein disulfide isomerase family, which promote correct protein folding via isomerization of disulfide bonds. The ER-60 is composed four domains, a, b, b’, and a’. Both a and a’ domain have active Cys-Gly-His-Cys (CGHC) motif. In each CGHC motif, two cysteines take either S-S (oxidized) or -SH (reduced) states. We have obtained SAXS profiles of ER-60 with both all CGHC oxidized (oxidized ER-60) and CGHC reduced (reduced ER-60). Our SAXS profiles did not match known crystal structure, and the SAXS profiles of the two states were slightly different from each other.

To investigate behavior of ER-60 in solution, we performed multi-scale molecular dynamics simulations. First, fluctuation of each domain was examined by atomistic MD simulations. The fluctuation around active site differed between oxidized and reduced ER-60, but no significant difference was seen in the other regions. It suggests that the difference of SAXS profile between two states is not due to the difference of intra-domain dynamics.

Second, motion of full-length ER-60 was examined by coarse-grained molecular dynamics (CGMD) simulations with CG Martini model, where each amino acid is represented by one to six particles. We have successfully obtained simulation trajectory which reproduce our SAXS profile. From the simulation trajectory, we analyzed inter-domain interface and frequency of binding/dissociation of each pair of the four domains.

Third, structural difference between oxidized ER-60 and reduced ER-60 was studied by coarser CGMD simulations with AICG2+ model, which enable extensive structural-sampling. We compared simulation snapshots which reproduce SAXS profile of oxidized ER-60 with simulation snapshots which reproduce that of reduced ER-60. Our simulation showed that the difference of two SAXS profiles reflect the difference in position of a’ domain.

Detergents are commonly used to disrupt noncovalent interactions of proteins, leading to detergent-protein complex or stabilized recombinant proteins. In past, many methods have been used to investigate conformational changes of proteins and protein-detergent complexes to understand their interactions, polarity and stability in varied detergent concentrations. The local structure of such protein/detergent complex could be resolved by spectroscopies; however, resolving the corresponding stoichiometric protein unfolding conformation requires separating the effects contributed by the coexisted protein/detergent complex and SDS micelles in the solution.

Figure 1. R_g and I₀ profiles extracted from the SAXS data measured along the chromatogram I_{UV280 nm} of the BSA/SDS solution in SDS buffer. Bottom shows the DAMMIF models of the complexes at the states indicated. (Shown models are assumed as single phase and further analysed by two-phase MD simulation.).

In this work, we show that sodium dodecyl sulfate (SDS), a frequently used surfactant in purification of membrane proteins, can bind to bovine serum albumin (BSA) for multistage unfolding. The on-line protein purification system of high performance liquid chromatography (SEC-HPLC) incorporated to the beamline 13A synchrotron BioSWAN instrument of the Taiwan Photon Source at the National Synchrotron Radiation Research Center, allows separating the scattering contributions from the BSA/SDS complexes and SDS micelles. Together with integrated observations of UV-vis absorption and refractive index (RI), we have resolved the stoichiometric unfolding conformations of BSA by SDS monomers to micelles. Offline SEC-MALS (multi angle light scattering (MALS) results are also consistent. In Figure 1, the corresponding protein-SDS association numbers along the unfolding process are determined uniquely from a combined analysis of UV-Vis absorption, refractive index, and zero-angle SAXS intensity measured in one sample elution. WAXS features (not shown) revealed the conformational change of complex inter-domain motions.

A cataract is a common disease for the aged people and has a very high chance to lead blindness. Instead of the surgery of replacing the clouding eye lens with an artificial one, it’s important to develop a non-surgical therapy. But it’s difficult to be carried out due to the lack of understanding on mechanism of cataract.

In the vertebrate eye lens, alpha-crystallin(α-crystallin) is the major structural protein and consists of two subunits, αA and αB, which are used to maintain lens transparency throughout life. As a member of the small heat shock protein family (sHsp), α-crystallin exhibits chaperone-like activity to prevent misfolding as well as aggregation of key proteins in the lens associated with cataract diseases. The previous studies reported that binding capacity of α-crystallin to lens lipids increases with age, and high molecular complex, comprising α-crystallin and misfolding protein, showed higher association with membrane. Recent evidences showed that sterols compounds can improve lens transparency. Due to the strong interaction between sterols with membranes, we proposed a model based on the membrane-mediated sterol-crystallin interaction.

In this study, we used αA and αB crystallin proteins, ergosterol and membranes as a model system to study the interactions between proteins, sterol molecules, and membranes. First, the influence of membrane on chaperone-like activity of αA and αB were checked by the assays of insulin, lysozyme and alcohol dehydrogenase (ADH). Circular dichroism (CD) was used to monitor the secondary structure changes of crystallin proteins induced by binding to membranes. Lamellar X-ray diffraction (LXD) was used to probe crystallin-induced structural change of membranes. Furthermore, small-angle X-ray scattering (SAXS) was used to probe structural changes of membranes with and without ergosterol induced by protein binding. The effects of ergosterol on the interaction between crystallin proteins and membranes will be discussed.

Neural precursor cells expressed developmentally downregulated protein 4– 2 (Nedd4-2) plays a key role in the ubiquitination process, which leads to the endocytosis and degradation of its downstream target molecules such as membrane proteins. Nedd4-2 belongs to the HECT ubiquitin ligase family, which regulates signal transduction through interaction with other proteins including 14-3-3 proteins. 14-3-3s are evolutionarily conserved proteins, which negatively regulate Nedd4-2 in cAMP- dependent manner through phosphorylation by protein kinase A (PKA). This regulation is performed by providing scaffolding for Nedd4-2, thereby preventing the interaction with Nedd4-2 and other membrane proteins. Though this is known, the molecular mechanism of this regulation remains unknown and is under scientific scrutiny. We aim to understand the structural and functional basis of 14-3-3 mediated regulation of Nedd4-2 using combined structural biology and biophysical approaches such as fluorescence spectroscopy, protein crystallography and chemical crosslinking coupled with mass spectroscopy

Possible mechanism of the 14-3-3 mediated inhibition of pNedd4-2 includes stabilization of inactive conformation of Nedd4-2 in which, HECT and C2 domains are involved in the intramolecular interaction and steric masking of WW domains surfaces. To test this hypothesis, we performed the time resolved fluorescence spectroscopy measurements using phosphorylated Nedd4-2 variants labelled by extrinsic fluorophore and monitor their interaction with 14-3-3 protein. Fluorescence spectroscopy provided basic information on the dynamics of the interaction between Nedd4-2 ligase and 14-3-3 protein. Measuring of rotational correlation time and determination of the mean lifetime values of excited fluorophore in Nedd4-2 alone and in the complex with 14-3-3 protein allow us to trace the microenvironment of one particular cysteine ​​amino acid, which is located at different positions within Nedd4-2 construct.

We also crystallized the complex of 14-3-3γΔC with the peptide containing phosphorylated Ser³⁴², solved its structure using molecular replacement and refined it at 1.61 Å resolution.

1. J. A. Manning and S. Kumar, Trends Biochem. Sci. 43, (2018), 635–647.

2. P. Goel, J. A. Manning, and S. Kumar, Gene, 557, (2015), 1–10.

3. Nagaki K, Yamamura H, Shimada S, Saito T, Hisanaga S, Taoka M, Isobe T, Ichimura T,

Biochemistry, 45, (2006), 6733-40.

4. Ichimura T, Isobe T, J Biol Chem., 280, (2005), 13187-94.

This study was supported by the Czech Science Foundation (Projects 20-00058S), the Czech Academy of Sciences (Research Projects RVO: 67985823 of the Institute of Physiology) and by Grant Agency of Charles University (Project No.348421).

The TIR superfamily includes membrane receptors, Interleukin-1 receptors (IL-1Rs) and Toll-like receptors (TLRs) and also TIR-containing cytoplasmic adaptor proteins such as MAL and MyD88. These proteins play a major role in immune signalling and are vital to innate host defense, inflammation, injury and stress [1]. IL-1R8, also known as single immunoglobulin interleukin-1 receptor-related protein (SIGIRR) is an inhibitory receptor from IL-1R family which regulates signalling of both IL-1Rs and TLRs. The mechanism of inhibition is not yet known, but the only available genetic evidence suggests that the conserved intracellular TIR domain of IL-1R8 alone is necessary to inhibit LPS-induced TLR4 signalling [2]. The recent cryo-EM structure of the MAL protofilament has revealed the molecular mechanism of TIR-TIR interactions in the MAL and MyD88 dependent TLR4 signalling [3]. Based on this, we hypothesize that a similar TIR:TIR interaction between the TIR domain of IL-1R8 and the TIR domains of either TLR4/MAL/MyD88 would be involved in the inhibition mechanism.

The TIR domain of human IL-1R8 was cloned, expressed and purified using E. coli host system. Turbidity assays, negative-stain electron microscopy (EM) and single-molecule fluorescence spectroscopy (SMFS) analysis indicated a potential interaction between IL-1R8^TIR and MAL^TIR. MAL^TIR forms filamentous assemblies when incubated with IL-1R8^TIR (Fig. 1). We are currently focusing on solving the 3D structure of MAL^TIR/IL-1R8^TIR filaments using negative-stain EM and cryo-EM to obtain molecular insights into the interaction interfaces and binding sites of IL-1R8^TIR and MAL^TIR. This study will eventually lead to an understanding of how TLR4 signalling is regulated by IL-1R8 and can potentially pave way in development of new therapeutic agents in future.

Figure 1. Left: Model representing the inhibition of TLR4 signalling by IL-1R8. Right: Negative-stain EM image of MAL^TIR/IL-1R8^TIR filaments taken using Hitachi HT 7700 TEM.

[1] Boraschi, D. et al. (2018). Immunol Rev. 281, 97-232[2]. Qin, J. et al. (2005). J Biol Chem. 280, 25233-25241[3]. Ve, T. et al. (2017). Nat. Struc. Mol. Biol. 24, 743-751

Paratellurite TeO₂ crystals under the application of a strong electric field demonstrate significant changes of the shape of allowed reflections, which are associated with the migration of oxygen vacancies to the surface layers [1]. Similar effect was found earlier in strontium titanate SrTiO₃ and got the name of “migration-induced field-stabilized polar phase” [2].

An experiment was carried out at P23 beamline of PETRA III synchrotron, devoted to the study of the changes in the forbidden reflections 002 and 100 in TeO₂ under applied electric field. These reflections are forbidden in conventional X-ray scattering, but can be observed at the energies close to absorption L-edges of Te, due to appearance of dipole-dipole resonant contribution to the atomic factor of Te. The experiment was carried out at the incident radiation energy, close to L₁ edge of Te 4938 eV. For both reflections the azimuthal dependence and energy spectrum were measured with and without application of electric field. For 002 reflection electric field magnitude was 500 and 750 V/mm, for 100 reflection it was 750 and 1050 V/mm.

We have observed a change of azimuthal dependence (Fig.1) caused by the violation of a symmetry in electric field in accordance with the predictions of preliminary theoretical calculations. Also we have observed a change of the energy spectrum at the field magnitude of 500 V/mm. It is assumed that this change is caused by appearance of oxygen vacancies in the environment of Te. For reflection 100 this change of the energy spectrum was even more obvious. This is justified because in this experimental geometry migration of vacancies is more pronounced.

[1] A. G. Kulikov, A. E. Blagov, N. V. Marchenkov, et.al. // JETP Letters, 107:10 (2018), 646–650 [2] J.Hanzig, M.Zschornak, F.Hanzig, et.al // Physical Review B 88, 024104 (2013)

A new biological small-angle X-ray scattering (BioSAXS) beamline is developed with the 3.0 GeV Taiwan Photon Source (TPS), for studies of biological structures in a wide range of length and time scales. The beamline provides a high flux (4 x10¹⁴ photons/s) for time-resolved and synchronized small- and wide-angle X-ray scattering (SAXS-WAXS), and offers new opportunities for ultra-SAXS (USAXS) to resolve the hierarchical structures of bio-machinery assemblies in solution, gel or condensed forms and anomalous SAXS/WAXS for metal or mineral distributions and compositions in an organelle or drug carrier. The beamline application extends to microbeam SAXS/WAXS for correlated crystal and nanostructural mappings in natural fibril tissues and synthetic biomaterials under tailored environmental controls. Concomitant SAXS-WAXS data collections are realized with a unique detecting system comprising an Eiger X-9M detector for SAXS and a custom-designed Eiger X-1M detector for simultaneous WAXS. These two X-ray detectors (75 mm pixel resolution) move independently with multi-degrees of freedom inside a large vacuum vessel of 12 m long and 1.5 m dia., providing dynamic and fast changes in detecting configuration for optimized data collections. Solution SAXS and WAXS of biomacromolecules are facilitated with an integrated system of online sample purification system of HPLC, strengthened by onsite UV-vis absorption followed by refractive of index measurement in one sample elution. The beamline has been opened to users since September 2020.

The Centre of Molecular Structure (CMS) provides services and access to state-of-art instruments, which cover a wide range of techniques required by not only structural biologists. CMS operates as part of the Czech Infrastructure for Integrative Structural Biology (CIISB), and European infrastructures Instruct-ERIC and MOSBRI. CMS is organized in 5 core facilities: CF Protein Production , CF Biophysics, CF Crystallization of proteins and nucleic acids, CF Diffraction techniques, and CF Structural Mass Spectrometry.

CF Diffraction techniques employs two laboratory X-ray instruments equipped with high flux MetalJet X-ray sources: a single crystal diffractometer D8 Venture (Bruker) and a small angle X-ray scattering instrument SAXSpoint 2.0 (Anton Paar). The configurations of both instruments represent top tier of possibilities of laboratory instrumentation. Apart from standard applications, the instruments are also extended for advanced experiments: the diffractometer is equipped with the stage for in-situ crystall diffraction and crystal dehydration, SAXS is equipped with in-situ UV-Vis spectroscopy and liquid chromatography system for SEC-SAXS. The setups enable easy access and fast turn-around of samples under different conditions, but also collection of high quality end-state data without further need for synchrotron data collection in many cases. CF Diffraction provides services in synergy with the other CFs on-site, therefore scientific questions can be quickly answered as they emerge from the experiments.

The Centre of Molecular Structure is supported by: MEYS CR (LM2018127); project Czech Infrastructure for Integrative Structural Biology for Human Health (CZ.02.1.01/0.0/0.0/16_013/0001776) from the ERDF; UP CIISB (CZ.02.1.01/0.0/0.0/18_046/0015974), and ELIBIO (CZ.02.1.01/0.0/0.0/15_003/0000447).

Type I collagen solution (bovine skin based) is studied using the biological small- and wide-angle X-ray scattering beamline at the 3.0 GeV Taiwan Photon Source of the National Synchrotron Radiation Research Center. Concomitant SAXS-WAXS data are collected from the sample elution with an online size exclusion column (SEC) of HPLC, incorporated with UV-vis absorption followed by refractive of index measurements. SEC-SAXS result indicates a relatively monodisperse size distribution of the tropocollagen, which comprises three left-handed helices of polypeptide strands that are twisted together into a right-handed coiled coil for a triple helix. The SAXS-revealed gyration R_g of 195 Å and elongated shape together with the molecular mass and the hydrodynamic radius R_h measured from dynamic light scattering and multi-angle laser light scattering, together, indicate a dimer form of the tropocollagen. Interestingly, these dimers can gradually form visible networks in solution upon adding short peptides; further, circular dichroism result indicates that these peptides are fond to reserve better the secondary structure of tropocollagen in solution upon UV illumination. The network formation mechanism of tropocollagen will be discussed in terms of the interaction of tropocollagen with the short peptides.