Structural characterization of proteins and complexes using small-angle X-ray solution scattering

Abstract

Small-angle scattering of X-rays (SAXS) is an established method for the low-resolution structural characterization of biological macromolecules in solution. The technique provides three-dimensional low-resolution structures, using ab initio and rigid body modeling, and allow one to assess the oligomeric state of proteins and protein complexes. In addition, SAXS is a powerful tool for structure validation and the quantitative analysis of flexible systems, and is highly complementary to the high resolution methods of X-ray crystallography and NMR. At present, SAXS analysis methods have reached an advanced state, allowing for automated and rapid characterization of protein solutions in terms of low-resolution models, quaternary structure and oligomeric composition. In this communication, main approaches to the characterization of proteins and protein complexes using SAXS are reviewed. The tools for the analysis of proteins in solution are presented, and the impact that these tools have made in modern structural biology is discussed.

Introduction

Small-angle scattering (SAS) of X-rays (SAXS) and neutrons (SANS) is a powerful method for the analysis of biological macromolecules in solution. Great progress has been made over the years in applying this technique to extract structural information from non-crystalline samples in the fields of physics, materials science and biology (Feigin and Svergun, 1987). Over the last decade, major advances in instrumentation and computational methods have led to new and exciting developments in the application of SAXS to structural biology. Active research is now being conducted by an increasing number of laboratories on advancing ab initio and rigid body modeling methods, the calculation of theoretical scattering curves from atomic models and the characterization of quaternary structure and intrinsic flexibility. In addition, advances in the automation of data collection and analysis make high throughput applications of SAXS experiments tractable (Hura et al., 2009, Round et al., 2008). Such developments have generated a renewed interest in the wider applications of the technique in the structural biology community. The present review is focused on the characterization of protein structure and complex formation, but the method is widely used for other macromolecular structures, e.g. RNA (Doniach and Lipfert, 2009, Rambo and Tainer, 2010).

Production of good quality samples is a prerequisite for a successful structural study by any method, and modern approaches to protein expression and purification used in structural biology laboratories help to facilitate this. Like the high resolution methods X-ray crystallography and nuclear magnetic resonance (NMR), SAS requires milligram amounts of highly pure, monodisperse protein that remains soluble at high concentration. However, while sample requirements are similar for the three methods (noting that an additional crystallization step is not required for the solution methods) a distinct advantage of SAXS is the speed of both data collection and sample characterization. On a modern synchrotron, scattering data can be collected in seconds, allowing an almost immediate characterization of the sample and the sample quality through the extraction of several overall parameters from the radially averaged scattering pattern. SAXS can thus be used as a method for the rapid screening of samples in various aqueous solvents/additives, including e.g. identification and optimization of crystallization conditions (Bonnete et al., 1999, Hamiaux et al., 2000).

In this review the discussion of SAS focusses on the elastic scattering of X-rays, SAXS, where dissolved macromolecules are exposed to a collimated and (for synchrotrons) focussed X-ray beam and the scattered intensity I is recorded by a detector as a function of the scattering angle (Fig. 1A). For an in-depth review of the theory behind SAXS the reader is directed to text-books and recent reviews (Feigin and Svergun, 1987, Koch et al., 2003, Putnam et al., 2007, Svergun, 2007, Svergun and Koch, 2003, Tsuruta and Irving, 2008), here some of the basic concepts will be presented with a focus on the characterization of proteins and protein complexes.

Scattering of X-rays by a solution of biomolecules is dependant on the number of biomolecules in the illuminated volume (i.e. to the solute concentration) and the excess scattering length density (often also called the contrast). For X-rays, the excess scattering length density, Δρ(r), comes from the difference in the electron density of the solute and solvent which, for biomolecules in aqueous solutions is very small. Consequently, synchrotron SAXS beamlines and laboratory sources must be optimized for the minimization of the contribution of background.

Dilute aqueous solutions of proteins, nucleic acids or other macromolecules give rise to an isotropic scattering intensity, which depends on the modulus of the momentum transfer s (s = 4πsin(θ)/λ, where 2θ is the angle between the incident and scattered beam):

$$I(s)=〈\mathbf{I}(\mathbf{s}){〉}_{\Omega }=〈\mathbf{A}(\mathbf{s}){\mathbf{A}}^{\ast }(\mathbf{s}){〉}_{\Omega }$$

where the scattering amplitude A(s) is a Fourier transformation of the excess scattering length density, and the scattering intensity is average over all orientations (Ω). Following subtraction of the solvent scattering, the background corrected intensity I(s) is proportional to the scattering of a single particle averaged over all orientations.

The scattering patterns generated from a dilute solution of macromolecules are typically presented as radially averaged one-dimensional curves (Fig. 1B). From these curves several overall important parameters can be directly obtained providing information about the size, oligomeric state and overall shape of the molecule. However, advances in computational methods have now made it possible to not only extract these simple parameters, but to also determine reliable three-dimensional structures from scattering data. Low-resolution (1–2 nm) SAXS models can be determined ab initio or through the refinement of available high-resolution structures and/or homology models. While the former analysis provides a low-resolution shape of the molecule in question and often adds insight to the biological problem at hand, the latter combination of SAXS and complementary data is a powerful method for the determination of the organisation of macromolecular complexes. In addition to structure determination SAXS is routinely used for the validation of structural models, the quantitative analysis of oligomeric state and the estimation of volume fractions of components in mixtures/polydisperse systems. While SAXS has been readily employed for the analysis of flexible systems including solutions of intrinsically unfolded proteins, methods were often restricted to the determination of simple geometric parameters. A renaissance in the study of such systems by structural biologists over the last 5–10 years has led to the development of novel approaches for the analysis of flexible systems including multi-domain and intrinsically unfolded proteins (Bernado et al., 2005, Bernadó et al., 2007, Obolensky et al., 2007).

SAXS is a technique that can probe structure on an extremely broad range of macromolecular sizes (Feigin and Svergun, 1987). Small proteins and polypeptides in the range of 1–10 kDa, macromolecular complexes and large viral particles up to several hundred MDa can all be measured with modern instrumentation under near native conditions. It is often attractive to laboratory based researchers as the amount of material required for a complete study is relatively low (typically 1–2 mg protein), and almost any biologically relevant sample conditions can be used. The effect of changes to sample environment (pH, temperature, salt concentration and ligand/co-factor titration) can be easily measured and, moreover, at high-brilliance synchrotron beamlines time-resolved experiments can be conducted (Lamb et al., 2008a, Lamb et al., 2008b, Pollack and Doniach, 2009, West et al., 2008).

It should be noted here that the elastic scattering of neutrons (SANS) is also widely used to characterize macromolecular solutions. Moreover, many approaches described below for SAXS are also applicable for SANS, where the excess scattering length density (contrast) is due to the nuclear (and sometimes spin) scattering length density instead of the electron density. In SANS, samples highly absorbing to X-rays (e.g. solvents containing high salt) can be measured, and the samples will not suffer from radiation damage. Most importantly, contrast variation by hydrogen/deuterium exchange can be used yielding precious additional information about the structure of macromolecular complexes. The disadvantages to SANS are that it usually requires more material than is required for SAXS, buffer subtraction is often difficult due to the high incoherent hydrogen scattering and that the measurements cannot be done on a laboratory source. Overall, SANS is a powerful complementary tool to SAXS (Ibel and Stuhrmann, 1975, Petoukhov and Svergun, 2006, Wall et al., 2000, Whitten and Trewhella, 2009).