For this complete IgG1 starting structure, force field parameterizations were generated and hydrogen atoms added using the glycan reader component of CHARMM-GUI (37,38) and the CHARMM36 force field (39, 40, 41, 42). and effector-binding Fc regions. Starting from 231,492 and 190,437 acceptable conformations for IgG1 and IgG4, respectively, joint x-ray and neutron scattering curve fits gave low goodness-of-fit factors for 28 IgG1 and 2748 IgG4 structures that satisfied the disulphide connectivity in their hinges. These joint best-fit structures showed that the best-fit IgG1 models had a greater separation between the centers of their Fab regions than those for IgG4, in agreement with their hinge lengths of 15 and 12 residues, respectively. The resulting asymmetric IgG1 solution structures resembled its crystal structure. Both symmetric and asymmetric solution structures were determined for IgG4. Docking simulations with our best-fit IgG4 structures showed greater steric clashes with its receptor to explain its weaker Fcand dihedral angles rapidly generated further models in which models with poor steric overlaps were discarded at the point of generation. These were fitted against scattering data. SASSIE was used to determine a solution structure for human monoclonal IgG2 from SANS data (13). Here, to validate this method in greater detail, SASSIE was used to remodel our previous joint SAXS and SANS data sets for?human monoclonal IgG1 and IgG4 (15,16) alongside new joint IgG1 and IgG4 scattering data from three other instruments to test their experimental reproducibility. Initial structures for full-length IgG1 and IgG4 were thus created using molecular dynamics. Monte Carlo simulations rapidly generated 700, 000 physically realistic IgG1 and IgG4 trial models to explore the physically allowed conformational space around the hinge region. Joint filters based on disulphide bridge constraints and consistency with joint SAXS and SANS fits resulted in a limited number of new atomistic solution structures that reflected the different hinge lengths of IgG1 and IgG4. The resulting models confirmed in greater detail and rigor our previous SCT/SCTPL analyses of IgG1 and IgG4 (15,16). Docking studies of these best-fit full-length IgG structures with their receptors provided, to our knowledge, new insight into the different functional roles of IgG1 and IgG4; therefore, the use of SASSIE is able to enhance our understanding of antibody-receptor-binding function. Materials and Methods Source of IgG1 and IgG4 antibodies The monoclonal IgG1 6a and IgG1 19a antibodies were used here as previously described (16). The monoclonal IgG4 B72.3 antibody in its Ser222 Eugenol and Pro222 forms was also used as previously described, with the Ser222 form corresponding to the wild-type hinge and the Pro222 form having a stabilized IgG4 hinge structure that prevents Fab-arm exchange (15). For new data collection, both IgG1 and IgG4 antibodies were purified by size-exclusion chromatography using a Superose 6 10/300 column (GE Healthcare, Chicago, IL) to remove nonspecific aggregates immediately before the SAXS and SANS measurements. For x-ray work, all four antibodies were measured in phosphate-buffered saline (PBS)-137 (137?mM NaCl, 8.1?mM Na2HPO4, 2.7?mM KCl, 1.5?mM KH2PO4 (pH 7.4)), and replacing 137?mM NaCl with 50?mM NaCl or 250?mM NaCl in buffers termed PBS-50 and PBS-250. Eugenol For neutron work, the light water in PBS-137 was replaced by 100% heavy water by extensive dialysis into 100% NF-ATC heavy water immediately before SANS experiments. By this, the H atoms in the hydration shell are replaced by D atoms, together with 90% of the protein-exchangeable H-atom content (18). Additional data collection utilized Hepes-137 buffer (10?mM Eugenol Hepes, 137?mM NaCl, and 2?mM CaCl2 (pH 7.4)). SAXS and SANS data for IgG1 and IgG4 Our previous SAXS data for IgG1 6a and 19a and IgG4 B72.3 in PBS-50, PBS-137, and PBS-250 buffers (15,16) were recorded in 16-bunch mode on instrument ID02 at the European Synchrotron Radiation Facility, Grenoble, France (24). This used a detector with a resolution of 512? 512 pixels in sets of 10 time frames with exposure times of 0.1 or 0.2?s each. New SAXS data (experimental ID: MX1801) were acquired using the BioSAXS robot on instrument BM29 at the European Synchrotron Radiation Facility (25,26). Data collection utilized a CMOS hybrid pixel Pilatus 1M detector (Dectris, Baden, Switzerland) with a resolution.
