Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution
Summary
The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.
Abstract
In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 – 5°) and wide angles (typically > 5°). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg’s law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.
Protocol
Representative Results
Discussion
The comparative procedure of the SWAXS system allows for numerous variables to be determined from experimental analysis. The parameters that are attained from the analysis can be used for different purposes according to the sample and experimental setup. SAXS provides information about nano-scale size and shape of the object, whereas WAXS focuses on the atomic and micro-scale structure (e.g. molecular lattice, unit cell dimension symmetry). More specifically, for particles in dilute solutions, SAXS can st…
Declarações
The authors have nothing to disclose.
Acknowledgements
We would like to thank Dr. Manfred Kriechbaum of Hecus XRS and the Institute of Biophysics and Nanosystems Research at the Austrian Academy of Sciences in Graz, Austria. LL and XW were supported in part by U.S. Department of Energy, under NERI-C Award No. DE-FG07-07ID14889, and U.S. Nuclear Regulatory Commission, under Award No. NRC-38-08-950. The SWAXS instrument is also supported in part by U.S. Department of Energy, under Award No. DE-NE0000325.
Materials
| Name of the products | Company |
| The System3 Small- and Wide-Angle X-Ray Scattering (SWAXS) Camera | Hecus XRS and IBN, Graz, Austria |
| GNOM | ATSAS 2.3 package by D. Svergun EMBL-Hamburg |
Referências
- Bernadó, P., Blackledge, M. Structural biology: Proteins in dynamic equilibrium. Nature. 468, 1046-1048 (2010).
- Zhang, F., Skoda, M. W. A., Jacobs, R. M. J., Martin, R. A., Martin, C. M., Schreiber, F. Protein Interactions Studied by SAXS: Effect of Ionic Strength and Protein Concentration for BSA in Aqueous Solutions. J. Phys. Chem. B. 111, 251-259 (2007).
- Maranas, J. K. The effect of environment on local dynamics of macromolecules. Current Opinion in Colloid & Interface Science. 12, 29-42 (2007).
- Stribeck, N. . X-Ray Scattering of Soft Matter. , (2007).
- Mertens, H. D., Svergun, D. I. Structural characterization of proteins and complexes using small-angle X-ray solution scattering. J. Struct. Biol. 172 (1), 128 (2010).
- Svergun, D. I. Small-angle X-ray and neutron scattering as a tool for structural systems biology. Biol. Chem. 391 (7), 737 (2010).
- Putnam, C. D., Hammel, M., Hura, G. L., Tainer, J. A. X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Quat. Rev. Biophys. 40, 191-285 (2007).
- Bonini, M., Fratini, E., Baglioni, P. SAXS study of chain-like structures formed by magnetic nanoparticles. Materials Science & Engineering C-Biomimetic and Supramolecular Systems. 27 (5-8), 1377-1381 (2007).
- Falletta, E., Ridi, F., Fratini, E., Vannucci, C., Canton, P., Bianchi, S., Castelvetro, V., Baglioni, P. A tri-block copolymer templated synthesis of gold nanostructures. Journal of Colloid and Interface Science. 357 (1), 88-94 (2011).
- Glatter, O., Scherf, G., Schillen, K., Brown, W. Characterization of a Poly(ethylene oxide) Poly(propylene oxide) Triblock Copolymer (EO(27)-PO39-EO(27)) in Aqueous-Solution. Macromolecules. 27 (21), 6046-6054 (1994).
- Mittelbach, R., Glatter, O. Direct structure analysis of small-angle scattering data from polydisperse colloidal particles. Journal of Applied Crystallography. 31, 600-608 (1998).
