Structural Characterization of Biomaterials by Means of Small Angle X-rays and Neutron Scattering (SAXS and SANS), and Light Scattering Experiments.

Scattering techniques represent non-invasive experimental approaches and powerful tools for the investigation of structure and conformation of biomaterial systems in a wide range of distances, ranging from the nanometric to micrometric scale. More specifically, small-angle X-rays and neutron scattering and light scattering techniques represent well-established experimental techniques for the investigation of the structural properties of biomaterials and, through the use of suitable models, they allow to study and mimic various biological systems under physiologically relevant conditions. They provide the ensemble averaged (and then statistically relevant) information under in situ and operando conditions, and represent useful tools complementary to the various traditional imaging techniques that, on the contrary, reveal more local structural information. Together with the classical structure characterization approaches, we introduce the basic concepts that make it possible to examine inter-particles interactions, and to study the growth processes and conformational changes in nanostructures, which have become increasingly relevant for an accurate understanding and prediction of various mechanisms in the fields of biotechnology and nanotechnology. The upgrade of the various scattering techniques, such as the contrast variation or time resolved experiments, offers unique opportunities to study the nano- and mesoscopic structure and their evolution with time in a way not accessible by other techniques. For this reason, highly performant instruments are installed at most of the facility research centers worldwide. These new insights allow to largely ameliorate the control of (chemico-physical and biologic) processes of complex (bio-)materials at the molecular length scales, and open a full potential for the development and engineering of a variety of nano-scale biomaterials for advanced applications.

Structural Characterization of Self-Assembling Hybrid Nanoparticles for Bisphosphonate Delivery in Tumors.

Hybrid self-assembling nanoparticles (hsaNPs) encapsulating bisphosphonates (BPs) recently showed very promising results in preclinic experiments for the treatment of brain tumor. However, the poor knowledge on the architecture of hybrid nanovectors is certainly one of the main reasons hampering further clinical and industrial development of these technologies. Here we propose to combine different techniques, that is, small angle neutron scattering (SANS) and X-ray Sscattering (SAXS), with cryo-electron transmission microscopy (cryo-TEM) to study the architecture of the final hsaNPs as well as of the four components before the assembling process. Data analysis based on SANS and SAXS experiments suggested a multiple compartment architecture of the final product, consisting of two bilayers sourrounding a core. Structures consisting of two shells surrounding an internal core were also observed in the cryo-TEM analysis. Such high resolution insight, also combined with size distribution and zeta potential of the NPs, provides exhaustive characterization of hsaNPs encapsulating BPs, and it is aimed at supporting further their clinical and industrial development.

Solution scattering approaches to dynamical ordering in biomolecular systems.

Clarification of solution structure and its modulation in proteins and protein complexes is crucially important to understand dynamical ordering in macromolecular systems. Small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS) are among the most powerful techniques to derive structural information. Recent progress in sample preparation, instruments and software analysis is opening up a new era for small-angle scattering. In this review, recent progress and trends of SAXS and SANS are introduced from the point of view of instrumentation and analysis, touching on general features and standard methods of small-angle scattering. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.

Inelastic and quasi-elastic neutron scattering spectrometers in J-PARC.

J-PARC, Japan Proton Accelerator Research Complex provides short pulse proton beam at a repetition rate 25Hz and the maximum power is expected to be 1MW. Materials and Life Science Experimental Facility (MLF) has 23 neutron beam ports and 21 instruments have already been operated or under construction/commissioning. There are 6 inelastic/quasi-elastic neutron scattering spectrometers and the complementary use of these spectrometers will open new insight for life science. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

What Can We Learn from Wide-Angle Solution Scattering?

Extending collection of x-ray solution scattering data into the wide-angle regime (WAXS) can provide information not readily extracted from small angle (SAXS) data. It is possible to accurately predict WAXS scattering on the basis of atomic coordinate sets and thus use it as a means of testing molecular models constructed on the basis of crystallography, molecular dynamics (MD), cryo-electron microscopy or ab initio modeling. WAXS data may provide insights into the secondary, tertiary and quaternary structural organization of macromolecules. It can provide information on protein folding and unfolding beyond that attainable from SAXS data. It is particularly sensitive to structural fluctuations in macromolecules and can be used to generate information about the conformational make up of ensembles of structures co-existing in solution. Novel approaches to modeling of structural fluctuations can provide information on the spatial extent of large-scale structural fluctuations that are difficult to obtain by other means. Direct comparison with the results of MD simulations are becoming possible. Because it is particularly sensitive to small changes in structure and flexibility it provides unique capabilities for the screening of ligand libraries for detection of functional interactions. WAXS thereby provides an important extension of SAXS that can generate structural and dynamic information complementary to that obtainable by other biophysical techniques.

Small Angle Scattering: Historical Perspective and Future Outlook.

Small angle scattering (SAS) is a powerful and versatile tool to elucidate the structure of matter at the nanometer scale. Recently, the technique has seen a tremendous growth of applications in the field of structural molecular biology. Its origins however date back to almost a century ago and even though the methods potential for studying biological macromolecules was realized already early on, it was only during the last two decades that SAS gradually became a major experimental technique for the structural biologist. This rise in popularity and application was driven by the concurrence of different key factors such as the increased accessibility to high quality SAS instruments enabled by the growing number of synchrotron facilities and neutron sources established around the world, the emerging need of the structural biology community to study large multi-domain complexes and flexible systems that are hard to crystalize, and in particular the development and availability of data analysis software together with the overall access to computational resources powerful enough to run them. Today, SAS is an established and widely used tool for structural studies on bio-macromolecules. Given the potential offered by the next generation X-ray and neutron sources as well as the development of new, innovative approaches to collect and analyze solution scattering data, the application of SAS in the field of structural molecular biology will certainly continue to thrive in the years to come.

Designing and Performing Biological Solution Small-Angle Neutron Scattering Contrast Variation Experiments on Multi-component Assemblies.

Solution small-angle neutron scattering (SANS) combined with contrast variation provides information about the size and shape of individual components of a multi-component biological assembly, as well as the spatial arrangements between the components. The large difference in the neutron scattering properties between hydrogen and deuterium is key to the method. Isotopic substitution of deuterium for some or all of the hydrogen in either the molecule or the solvent can greatly alter the scattering properties of the biological assembly, often with little or no change to its biochemical properties. Thus, SANS with contrast variation provides unique information not easily obtained using other experimental techniques.If used correctly, SANS with contrast variation is a powerful tool for determining the solution structure of multi-component biological assemblies. This chapter discusses the principles of SANS theory that are important for contrast variation, essential considerations for experiment design and execution, and the proper approach to data analysis and structure modeling. As sample quality is extremely important for a successful contrast variation experiment, sample issues that can affect the outcome of the experiment are discussed as well as procedures used to verify the sample quality. The described methodology is focused on two-component biological complexes. However, examples of its use for multi-component assemblies are also discussed.

Structural Characterization of Highly Flexible Proteins by Small-Angle Scattering.

Intrinsically Disordered Proteins (IDPs) are fundamental actors of biological processes. Their inherent plasticity facilitates very specialized tasks in cell regulation and signalling, and their malfunction is linked to severe pathologies. Understanding the functional role of disorder requires the structural characterization of IDPs and the complexes they form. Small-angle Scattering of X-rays (SAXS) and Neutrons (SANS) have notably contributed to this structural understanding. In this review we summarize the most relevant developments in the field of SAS studies of disordered proteins. Emphasis is given to ensemble methods and how SAS data can be combined with computational approaches or other biophysical information such as NMR. The unique capabilities of SAS enable its application to extremely challenging disordered systems such as low-complexity regions, amyloidogenic proteins and transient biomolecular complexes. This reinforces the fundamental role of SAS in the structural and dynamic characterization of this elusive family of proteins.

SAS-Based Structural Modelling and Model Validation.

Small angle scattering of X-rays (SAXS) and neutrons (SANS) is a structural technique to study disordered systems with chaotic orientations of scattering inhomogeneities at low resolution. An important example of such systems are solutions of biological macromolecules. Rapid development in the methodology for solution scattering data interpretation and model building during the last two decades brought the analysis far beyond the determination of just few overall structural parameters (which was the only possibility in the past) and ensured SAS a firm position in the methods palette of the modern life sciences. The advances in the methodology include ab initio approaches for shape and domain structure restoration from scattering curves without a priori structural knowledge, classification and validation of the models, evaluation of potential ambiguity associated with the reconstruction. In rigid body and hybrid modelling approaches, solution scattering is synergistically used with other structural techniques utilizing the complementary information such as atomic models of the components, intramolecular contacts, subunits orientations etc. for the reconstruction of complex systems. The usual requirement of the sample monodispersity has been loosed recently and the technique can now address such systems as weakly bound oligomers and transient complexes. These state-of-the-art methods are described together with the examples of their applications and the possible ways of post-processing of the models.

High Resolution Distance Distributions Determined by X-Ray and Neutron Scattering.

Measuring distances within or between macromolecules is necessary to understand the chemistry that biological systems uniquely enable. In performing their chemistry, biological macromolecules undergo structural changes over distances ranging from atomic to micrometer scales. X-ray and neutron scattering provide three key assets for tackling this challenge. First, they may be conducted on solutions where the macromolecules are free to sample the conformations that enable their chemistry. Second, there are few limitations on chemical environment for experiments. Third, the techniques can inform upon a wide range of distances at once. Thus scattering, particularly recorded at small angles (SAS), has been applied to a large variety of phenomenon. A challenge in interpreting scattering data is that the desired three dimensional distance information is averaged onto one dimension. Furthermore, the scales and variety of phenomenon interrogated have led to an assortment of functions that describe distances and changes thereof. Here we review scattering studies that characterize biological phenomenon at distances ranging from atomic to 50 nm. We also distinguish the distance distribution functions that are commonly used to describe results from these systems. With available X-ray and neutron scattering facilities, bringing the action that occurs at the atomic to the micrometer scale is now reasonably accessible. Notably, the combined distance and dynamic information recorded by SAS is frequently key to connecting structure to biological activity and to improve macromolecular design strategies and outcomes. We anticipate widespread utilization particularly in macromolecular engineering and time-resolved studies where many contrasting experiments are necessary for resolving chemical mechanisms through structural changes.

Small Angle Scattering for Pharmaceutical Applications: From Drugs to Drug Delivery Systems.

The sub-nanometer scale provided by small angle neutron and X-ray scattering is of special importance to pharmaceutical and biomedical investigators. As drug delivery devices become more functionalized and continue decreasing in size, the ability to elucidate details on size scales smaller than those available from optical techniques becomes extremely pertinent. Information gathered from small angle scattering therefore aids the endeavor of optimizing pharmaceutical efficacy at its most fundamental level. This chapter will provide some relevant examples of drug carrier technology and how small angle scattering (SAS) can be used to solve their mysteries. An emphasis on common first-step data treatments is provided which should help clarify the contents of scattering data to new researchers. Specific examples of pharmaceutically relevant research on novel systems and the role SAS plays in these studies will be discussed. This chapter provides an overview of the current applications of SAS in drug research and some practical considerations for selecting scattering techniques.

Hybrid Applications of Solution Scattering to Aid Structural Biology.

Biomolecular applications of solution X-ray and neutron scattering (SAXS and SANS, respectively) started in late 1960s - early 1970s but were relatively limited in their ability to provide a detailed structural picture and lagged behind what became the two primary methods of experimental structural biology - X-ray crystallography and NMR. However, improvements in both data analysis and instrumentation led to an explosive growth in the number of studies that used small-angle scattering (SAS) for investigation of macromolecular structure, often in combination with other biophysical techniques. Such hybrid applications are nowadays quickly becoming a norm whenever scattering data are used for two reasons. First, it is generally accepted that SAS data on their own cannot lead to a uniquely defined high-resolution structural model, creating a need for supplementing them with information from complementary techniques. Second, solution scattering data are frequently applied in situations when a method such NMR or X-ray crystallography cannot provide a satisfactory structural picture, which makes these additional restraints highly desirable. Maturation of the hybrid bio-SAS approaches brings to light new questions including completeness of the conformational space sampling, model validation, and data compatibility.

Applications of SANS to Study Membrane Protein Systems.

Small angle neutron scattering (SANS) is a powerful tool to obtain structural information on solubilized membrane proteins on the nanometer length-scale in complement to other structural biology techniques such as cryo-EM, NMR and SAXS. In combination with deuteration of components and/or contrast variation (H2O:D2O exchange in the buffer) SANS allows to separate structural information from the protein and the detergent/lipid parts in solution. After a short historical overview on results obtained by SANS on membrane protein systems, this book chapter introduces the basic theoretical principles of the technique as well as requirements on samples. The two introductory sections are followed by an illustration of the specific consequences of sample heterogeneity of solubilized membrane proteins in the presence of detergent/lipid molecules on the interpretation of structural information by using simple, geometric models. The next sections deal with more sophisticated modelling approaches including ab initio shape reconstructions and full-atomic models in the presence of detergent/lipid and specific results obtained by these approaches. After a short comparison with the SAXS technique, this book chapter concludes with an overview of present and future developments and impact that can be expected by SANS on membrane structural biology in the coming years.

Dual spectrum neutron radiography: identification of phase transitions between frozen and liquid water.

In this Letter, a new approach to distinguish liquid water and ice based on dual spectrum neutron radiography is presented. The distinction is based on arising differences between the cross section of water and ice in the cold energy range. As a significant portion of the energy spectrum of the ICON beam line at Paul Scherrer Institut is in the thermal energy range, no differences can be observed with the entire beam. Introducing a polycrystalline neutron filter (beryllium) inside the beam, neutrons above its cutoff energy are filtered out and the cold energy region is emphasized. Finally, a contrast of about 1.6% is obtained with our imaging setup between liquid water and ice. Based on this measurement concept, the temporal evolution of the aggregate state of water can be investigated without any prior knowledge of its thickness. Using this technique, we could unambiguously prove the production of supercooled water inside fuel cells with a direct measurement method.

A neutron reflection study of surface enrichment in nematic liquid crystals.

The interfacial adsorption properties of several different dopants in cyanobiphenyl liquid crystals have been measured using specular neutron reflection. It was found that a partly fluorinated analogue of 11OCB, called F17, adsorbed strongly at the interface between 5CB and air but it was not adsorbed at the interface between 5CB and a solid substrate treated with cetyl trimethyl ammonium bromide (CTAB). The concentration dependence of the adsorption at the air interface was well described by the Brunauer, Emmett and Teller (BET) model, adapted for solutions rather than the gas phase. The isotherms are determined by two equilibrium constants: K(S) for adsorption of the dopant directly at the interface and K(L) for adsorption onto previously adsorbed dopant. The temperature dependence of K(S) indicated that the adsorption enthalpy is not influenced by the phase of the 5CB and its value of -29 kJmol(-1) is consistent with physical adsorption. The value of K(L) is zero in the isotropic phase but increases rapidly on cooling in the nematic phase suggesting that the F17 is less compatible with nematic than isotropic 5CB. The smallest layer thicknesses (~18 Å) suggest that the F17 molecules are approximately perpendicular to the surface. The other dopants studied were components of the E7 mixture: 8OCB and 5CT. No adsorption was found for 8OCB but 5CT showed adsorption at a CTAB treated solid interface when present in 5CB at the 10% level. In this case, the value of K(S) was much smaller than for F17 but the value of K(L) was such that an exponential concentration profile (predicted by the BET model) was observed with characteristic thickness of ~200 Å. The results demonstrate the potential for very precise control of surface properties in liquid crystal devices by using appropriate dopants.

Neutron macromolecular crystallography with LADI-III.

At the Institut Laue-Langevin, a new neutron Laue diffractometer LADI-III has been fully operational since March 2007. LADI-III is dedicated to neutron macromolecular crystallography at medium to high resolution (2.5-1.5 Å) and is used to study key H atoms and water structure in macromolecular structures. An improved detector design and readout system has been incorporated so that a miniaturized reading head located inside the drum scans the image plate. From comparisons of neutron detection efficiency (DQE) with the original LADI-I instrument, the internal transfer of the image plates and readout system provides an approximately threefold gain in neutron detection. The improved performance of LADI-III, coupled with the use of perdeuterated biological samples, now allows the study of biological systems with crystal volumes of 0.1-0.2 mm(3), as illustrated here by the recent studies of type III antifreeze protein (AFP; 7 kDa). As the major bottleneck for neutron macromolecular studies has been the large crystal volumes required, these recent developments have led to an expansion of the field, extending the size and the complexity of the systems that can be studied and reducing the data-collection times required.

Neutron structure analysis using the IBARAKI biological crystal diffractometer (iBIX) at J-PARC.

The IBARAKI Biological Crystal Diffractometer (iBIX), a new diffractometer for protein crystallography at the next-generation neutron source at J-PARC (Japan Proton Accelerator Research Complex), has been constructed and has been operational since December 2008. Preliminary structure analyses of organic crystals showed that iBIX has high performance even at 120 kW operation and the first full data set is being collected from a protein crystal.

A history of neutrons in biology: the development of neutron protein crystallography at BNL and LANL.

The first neutron diffraction data were collected from crystals of myoglobin almost 42 years ago using a step-scan diffractometer with a single detector. Since then, major advances have been made in neutron sources, instrumentation and data collection and analysis, and in biochemistry. Fundamental discoveries about enzyme mechanisms, biological complex structures, protein hydration and H-atom positions have been and continue to be made using neutron diffraction. The promise of neutrons has not changed since the first crystal diffraction data were collected. Today, with the developments of beamlines at spallation neutron sources and the use of the Laue method for data collection, the field of neutrons in structural biology has renewed vitality.

[Beginning of use of a new biological neutron diffractometer (iBIX) in J-PARC].

Ibaraki Prefectural Government together with Ibaraki University and Japan Atomic Energy Agency (JAEA) has almost finished constructing a time-of-flight (TOF) neutron diffractometer for biological macromolecules for industrial use at J-PARC, IBARAKI Biological Crystal Diffractometer (iBIX). Since 2009, Ibaraki University has been asked to operate this machine in order for users to do experiments by Ibaraki Prefecture. The diffractometer is designed to cover sample crystals which have their cell edges up to around 150 A. It is expected to measure more than 100 samples per year if they have 2 mm(3) in crystal volume, and to measure even around 0.1 mm(3) in crystal volume of biological samples. The efficiency of iBIX is also expected about 100 times larger than those of the present high performance diffractometers at JRR-3 in JAEA when 1MW power realizes in J-PARC. Since December 2008, iBIX has been open to users and several proteins and organic compounds were tested under 20 kW proton power of J-PARC. It was found that one of their proteins was diffracted up to 1.4 A in d-spacing, which was nearly comparable resolution to that of BIX-3 in JRR-3 when used the same crystal as at iBIX for reasonable exposure time. In May 2009, 14 detector units were set up. By the end of fiscal year 2009, the basic part of data reduction software will be finished and an equipment blowing low temperature gas to the sample will be installed with the cooperation of JAEA.

[Collaborative use of neutron and X-ray for determination of drug target proteins].

Crystallography enables us to obtain accurate atomic positions within proteins. High resolution X-ray crystallography provides information for most of the atoms comprising a protein, with the exception of hydrogens. Neutron diffraction data can provide information of the location of hydrogen atoms, and is complementary to the structural information determined by X-ray crystallography. Here, we show the recent result of the structural determination of drug-target proteins, porcine pancreatic elastase and human immuno-deficiency virus type-1 protease by both X-ray and neutron diffraction. The structure of porcine pancreatic elastase with its potent inhibitor was determined to 0.94 A resolution by X-ray diffraction and 1.65 A resolution by neutron diffraction. The structure of HIV-PR with its potent inhibitor was also determined to 0.93 A resolution by X-ray diffraction and 1.9 A resolution by neutron diffraction. The ionization state and the location of hydrogen atoms of the catalytic residue in these enzymes were determined by neutron diffraction. Furthermore, collaborative use of both X-ray and neutron to identify the location of ambiguous hydrogen atoms will be shown.