Section 19.3.3.4.1. Studies of proteins in solution that complement high-resolution structures

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).