X-ray and neutron solution scattering methods provide multiparameter structural and compositional information on proteins that complements high-resolution protein crystallography and NMR studies. We describe the procedures required to (1) obtain validated X-ray and neutron scattering data, (2) perform Guinier analyses of the scattering data to extract the radius of gyration R(G) and intensity parameters, and (3) calculate the distance distribution function P(r). Constrained modeling is important because this confirms the experimental data analysis and produces families of best-fit molecular models for comparison with crystallography and NMR structures. The modeling procedures are described in terms of (4) generating appropriate starting models, (5) randomizing these for trial-and-error scattering fits, (6) identifying the final best-fit models, and (7) applying analytical ultracentrifugation (AUC) data to validate the scattering modeling. These procedures and pitfalls in them will be illustrated using work performed in the authors' laboratory on antibodies and the complement proteins of the human immune defense system. Four different types of modeling procedures are distinguished, depending on the number and type of domains in the protein. Examples when comparisons with crystallography and NMR structures are important are described. For multidomain proteins, it is often found that scattering provides essential evidence to validate or disprove a crystal structure. If a large protein cannot be crystallized, scattering provides the only means to obtain a structure.

Factor H (FH) is a plasma glycoprotein that plays a central role in regulation of the alternative pathway of complement. It is composed of 20 short complement regulator (SCR) domains. The SCR-1/5 fragment is required for decay acceleration and cofactor activity, while the SCR-16/20 fragment possesses binding sites for complement C3d and heparin. X-ray scattering and analytical ultracentrifugation showed that SCR-1/5 was monomeric, while SCR-16/20 formed dimers. The Guinier radius of gyration R(G) of 4.3 nm for SCR-1/5 and those of 4.7 nm and about 7.8 nm for monomeric and dimeric SCR-16/20, respectively, showed that their structures are partially folded back and bent. The distance distribution function P(r) showed that SCR-1/5 has a maximum dimension of 15 nm while monomeric and dimeric SCR-16/20 are 17 nm and about 27 nm long, respectively. The sedimentation coefficient of 2.4 S for SCR-1/5 showed no concentration-dependence, while that for SCR-16/20 was 2.8 S for the monomer and 3.9 S for the dimer. Sedimentation equilibrium data showed that SCR-1/5 is monomeric while SCR-16/20 exhibited a weak monomer-dimer equilibrium with a dissociation constant of 16 microM. The constrained scattering and sedimentation modelling of SCR-1/5 and SCR-16/20 showed that partially folded-back and bent flexible SCR arrangements fitted both data sets better than extended linear arrangements, and that the dimer was best modelled in the SCR-16/20 model by an end-to-end association of two SCR-20 domains. The SCR-1/5 and SCR-16/20 models were conformationally similar to the previously determined partially folded-back structure for intact wild-type FH, hence suggesting a partial explanation of the intact FH structure. Comparison of the SCR-16/20 model with the crystal structure of C3b clarified reasons for the distribution of mutations leading to atypical haemolytic uraemic syndrome.

Factor H (FH) is a major complement control protein in serum. The seventh short complement regulator (SCR-7) domain of the 20 in FH is associated with age-related macular degeneration through a Tyr402His polymorphism. The recombinant SCR-6/8 domains containing either His402 or Tyr402 and their complexes with a heparin decasaccharide were studied by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient is concentration dependent, giving a value of 2.0 S at zero concentration and a frictional ratio f/f(o) of 1.2 for both allotypes. The His402 allotype showed a slightly greater self-association than the Tyr402 allotype, and small amounts of dimeric SCR-6/8 were found for both allotypes in 50 mM, 137 mM and 250 mM NaCl buffers. Sedimentation equilibrium data were interpreted in terms of a monomer-dimer equilibrium with a dissociation constant of 40 microM for the His402 form. The Guinier radius of gyration R(G) of 3.1-3.3 nm and the R(G)/R(O) ratio of 2.0-2.1 showed that SCR-6/8 is relatively extended in solution. The distance distribution function P(r) showed a maximum dimension of 10 nm, which is less than the length expected for a linear domain arrangement. The constrained scattering and sedimentation modelling of FH SCR-6/8 showed that bent SCR arrangements fit the data better than linear arrangements. Previously identified heparin-binding residues were exposed on the outside curvature of this bent domain structure. Heparin caused the formation of a more linear structure, possibly by binding to residues in the linker. It was concluded that the His402 allotype may self-associate more readily than the Tyr402 allotype, SCR-6/8 is partly responsible for the folded-back structure of intact FH, and SCR-6/8 changes conformation upon heparin binding.

This chapter describes current knowledge on the ocular structure, function and barrier properties to drug delivery. A broad description of different ocular parts and their histology is provided. Different routes of drug administration such as topical, oral, intravenous, subconjunctival, periocular and intravitreal routes are employed to treat ocular ailments. Critical anatomical and physiological factors that regulate drug absorption are discussed. Of the drug administration routes mentioned, topical for anterior ocular tissues, and periocular and intravitreal for back of the eye tissues are mostly preferred and recommended. Ocular drug absorption is impeded by static, dynamic and metabolic barriers. Static ocular barriers include corneal epithelium, blood aqueous barrier, sclera, retinal pigment epithelium and blood capillary endothelial cells, whereas the dynamic ocular barriers include tear drainage, conjunctival blood and lymph clearance, and choroidal blood and lymphatic circulations. These barriers are segregated and described as anterior segment and posterior segment barriers. As a barrier, the role of ocular efflux transporters, which reduces intracellular xenobiotic concentration, is briefly described.

Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (±13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (±0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-α. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d–antigen complexes with the B cell receptor.

Complement receptor 2 (CR2; CD21) is a membrane-bound regulator of complement activation, being comprised of 15 or 16 short complement repeat (SCR) domains. A recombinant glycosylated human CR2 SCR 1-2 domain pair was engineered with the Fc fragment of a mouse IgG1 antibody to create a chimaera CR2-Ig containing the major ligand binding domains. Such a chimaera has therapeutic potential as a complement inhibitor or immune modulator. X-ray and neutron scattering and analytical ultracentrifugation identified its domain structure in solution, and provided a comparison with controversial folded-back crystal structures for deglycosylated CR2 SCR 1-2. The radius of gyration R(G) of CR2-Ig was determined to be 5.39(+/-0.14) nm and 5.29(+/-0.01) nm by X-ray and neutron scattering, respectively. The maximum dimension of CR2-Ig was determined to be 17 nm. The molecular mass of CR2-Ig ranged between 101,000 Da and 107,000 Da as determined by neutron scattering and sedimentation equilibrium, in good agreement with the sequence-derived value of 106,600 Da. Sedimentation velocity gave a sedimentation coefficient of 4.49(+/-0.11) S. Stereochemically complete models for CR2-Ig were constructed from crystal structures for the CR2 SCR 1-2 and mouse IgG1 Fc fragments. The two SCR domains and the Fc fragment were joined by randomised conformational peptides. The analysis of 35,000 possible CR2-Ig models showed that only those models in which the two SCR domains were arranged in an open V-shape in random orientations about the Fc fragment accounted for the scattering and sedimentation data. It was not possible to define one single conformational family of Fab-like fragment relative to the Fc fragment. This flexibility is attributed to the relatively long linker sequence and the absence of the antibody light chain from CR2-Ig. The modelling also confirmed that the structure of CR2 SCR 1-2 is more extended in solution than in its crystal structure.

Complement receptor type 2 (1033 aa, ~113 kDa) is encoded by the human CR2 gene. This protein plays a role in the activation of complement-mediated immunity.