International
Tables for Crystallography Volume F Crystallography of biological macromolecules Edited by M. G. Rossmann and E. Arnold © International Union of Crystallography 2006 |
International Tables for Crystallography (2006). Vol. F. ch. 19.3, pp. 436-437
Section 19.3.3.4. Recent applications of solution X-ray scattering in structural molecular biology
a
SSRL/SLAC & Department of Chemistry, Stanford University, PO Box 4349, MS69, Stanford, California 94309-0210, USA, and bDepartment of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA |
Solution X-ray scattering provides a direct means of measuring shape at low resolution, i.e., the radius of gyration, the maximum dimension within a protein and the relative molecular weight. Determining the oligomeric state of a protein system is straightforward. Dimensional parameters for simple objects can be obtained indirectly by fitting the observed pattern with spherical or higher-symmetry harmonics. The concept can be extended by use of partial structural models obtained by crystallography and NMR to construct a low-resolution model of large macromolecular complexes that are not amenable to these high-resolution techniques. The refinement of such models against the solution scattering data of the putative complex bridges between atomic resolution structures and biological functions performed by large complexes. Time-resolved studies expand knowledge derived by a variety of structural techniques into the context of experimental molecular dynamics. The examples given below demonstrate some such complementing aspects of solution X-ray scattering studies.
Recent advances in solution scattering data interpretation have made it possible to determine three-dimensional structures at resolutions comparable to those of electron microscopy. In some cases, low-resolution structures were derived from solution X-ray scattering independent of a structural model obtained by other structural techniques. Svergun et al. (1994) derived a 15 Å-resolution model of 50S ribosome using the spherical harmonics method. The low-resolution structures of the dimeric and tetrameric forms of pyruvate decarboxylase were obtained by the same method from X-ray solution scattering data (König et al., 1992) before the crystal structure was published. The X-ray structure, at a comparable resolution, was in close agreement with the solution scattering result. The same enzyme obtained from different organisms has also been modelled with solution scattering data.
Calcium-binding proteins have been extensively studied by solution X-ray scattering. These data first demonstrated the large conformation change of calmodulin promoted by binding Ca2+ (Seaton et al., 1985). Comparison of the solution scattering curve calculated for the crystal structure of calmodulin with the experimental scattering curve showed that crystal-packing forces substantially altered the solution conformation of the protein (Heidorn & Trewhella, 1988). Kataoka et al. (1989) reported the first glimpse of the conformational change of calmodulin induced by melittin, a model peptide for target enzymes. These early studies led to a number of important solution scattering studies on the protein–protein interaction in Ca2+-regulated molecular switching, including the troponin system in muscle contraction. Krueger et al. (1997) recently took a combined approach to X-ray and neutron small-angle scattering by using contrast variation to obtain the first structural model of calmodulin complexed with an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase.
A series of structural studies on 70 kDa heat-shock cognate protein, a molecular chaperone, combined crystallography and X-ray solution scattering. It was not possible to crystallize the whole protein, but the crystal structure of the ATPase domain was solved. ATP binding induces a conformational change in the protein, resulting in the release of a bound peptide. Wilbanks et al. (1995) constructed a low-resolution model of the whole molecule from solution X-ray scattering data, based on the ATPase domain crystal structure. Solution X-ray scattering was recently used to screen a point-mutated version of the protein, which retains the ATP-induced conformational change, contributing to the interpretation of the role of specific residues in the molecular chaperone mechanism (Sousa & McKay, 1998).
Solution scattering complemented high-resolution NMR structural studies in the investigation of titin (connectin), a giant muscle protein (Improta et al., 1998). The high-resolution structures were determined for two immunoglobulin-like fragments of the I-band region of titin. Two ellipsoids that simulate the molecular envelopes of the two fragments were then used to model solution X-ray scattering data in order to determine the relative position of the two fragments. The resulting structural model suggests that the motions around the interdomain-connecting regions are restricted and that titin behaves as a row of beads connected by rigid hinges. A similar approach was taken for EGF domains in coagulation factor X (Sunnerhagen et al., 1996).
There is a long list of large proteins or protein complexes whose solution X-ray scattering studies have made important contributions in structural biology. One study demonstrated that the quaternary structure of an allosteric enzyme, E. coli aspartate transcarbamoylase, in the R (relaxed) state is significantly different from that observed in the R-state crystal, suggesting that a crystal lattice could deform functional quaternary structure (Svergun, Barberato et al., 1997). A. vinelandii nitrogenase, the key enzyme in nitrogen fixation, is composed of two proteins: Fe protein and MoFe protein, which were crystallized separately. Grossman et al. (1997) determined a low-resolution structure of the complex of Fe and MoFe proteins stabilized by ADP–AlF4−, a nucleotide triphosphate analogue, solely from solution X-ray scattering data. This was later confirmed by a crystallographic study of the same complex (Schindelin et al., 1997). A more advanced approach was used to construct the three-dimensional structure of E. coli F1 ATPase, which consists of four different soluble subunits in the form of α3β3ɛδ (Svergun, Konrad et al., 1998). Two smaller subunits, ɛ and δ, were disordered in the crystal structure, while NMR provided solution structures for these subunits. Solution scattering was used to construct the three-dimensional structure of the F1 ATPase structure that incorporates all the subunits. The program CRYSOL was used to calculate individual scattering amplitudes by approximating high-resolution structures with sums of spherical harmonics. Then individual amplitudes were combined, according to the relative positions of all subunits, while the subunits were moved with respect to each other to fit the experimental scattering amplitudes obtained for the whole complex. The programs ASSA and ALM22INT were used to construct the most plausible model for the complex. A 32 Å-resolution structure of M. Sextra V1 ATPase was determined ab initio using the spherical harmonics method (Svergun, Aldag et al., 1998). Here, a three-fold molecular symmetry in the A3B3 part, which resembles α3β3 in F1 ATPase, reduced the number of parameters. The model structure of V1 ATPase has a 110 Å-long stalk region, corresponding to the total mass of the CDEFG3 part of the complex that connects V1 to the V0 domain embedded in the membrane.
X-ray scattering amplitudes have recently been used in the correction of the contrast-transfer function in cryo-electron microscopy in an attempt to improve the fidelity of reconstructed electron-microscope models (Thuman-Commike et al., 1999). A low-resolution structure model derived from solution X-ray scattering and electron microscopy was used to obtain initial phases for determining the crystal structure of E. coli ClpP, a molecular chaperone (Wang et al., 1997).
The advent of synchrotron-radiation sources made time-resolved X-ray studies of biological macromolecular systems possible. Time-resolved small-angle scattering studies on noncrystalline systems, such as skeletal muscle fibres and proteins in solution, are among the first studies of this type. There is still no other structural technique that offers the means of studying large, real-time conformational changes of macromolecular complexes that cannot be contained within a crystal lattice. This aspect of time-resolved solution scattering thus complements time-resolved Laue crystallography. Arguably, the most important contribution of small-angle solution scattering in recent years is the study of temporal changes in biological molecules resulting from changes in the solution environment. These changes are frequently induced by rapid mixing using a stopped-flow apparatus or a temperature jump that allows a virtually synchronous initiation of a reaction, so that the data reflect a common state for the vast majority of the molecules in solution. The changes may be associations, dissociations [e.g. the self-assembly of tobacco mosaic virus (Potschka et al., 1988) and microtubule assembly and oscillation behaviours (Mandelkow et al. 1989)], as well as changes in quaternary structure. In time-resolved studies of the allosteric transition of E. coli aspartate transcarbamoylase (Tsuruta et al., 1994), a structural intermediate during the enzyme reaction that differs from representative T and R quaternary structures was sought. As the technique has matured, a number of challenging studies have recently been conducted. Many of these time-resolved studies are focused on protein folding, as mentioned below.
There have been a number of solution X-ray scattering studies on protein folding in recent years, in which proteins of low molecular weight had to be investigated under low electron-density contrast due to the presence of denaturing agents, such as urea, at high concentration. Solution X-ray scattering complements other structural techniques used in protein-folding studies, as this is the only technique available for learning how compact a protein is in solution. A Kratky plot of solution scattering data serves as a quick means of determining whether a protein is folded or unfolded. Recently, the singular value decomposition method was applied to this class of problems and revealed a folding intermediate of lysozyme (Chen et al., 1996). This study has recently been expanded to a time-resolved study, which revealed a compact folding intermediate that has not yet formed a hydrophobic core (Chen et al., 1998). Similar studies are being carried out on other protein systems. Arai et al. (1998) reported that β-lactoglobulin undergoes a similar folding pattern while it forms a folding intermediate with a hydrophobic core within 100 ms. Uversky et al. (1998) reported association-induced folding of globular proteins. Pollack et al. (1999) developed a micro-machined mixer to study folding of cytochrome c in the sub-millisecond regime.
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