Epitope mapping the specific residues of an antibody/antigen interaction can be used to support mechanistic interpretation, antibody optimization, and epitope novelty assessment. Thus, there is a strong need for mapping methods, particularly integrative ones. Here we report identification of an energetic epitope by determining the interfacial hot-spot that dominates the binding affinity for an anti-IL-23 antibody by using the complementary approaches of hydrogen/deuterium exchange mass spectrometry (HDX-MS), fast photochemical oxidation of proteins (FPOP), alanine shave mutagenesis, and binding analytics. Five peptide regions on IL-23 with reduced backbone amide solvent accessibility upon antibody binding were identified by HDX-MS, and five different peptides over the same three regions were identified by FPOP. In addition, FPOP analysis at the residue-level reveals potentially key interacting residues. Mutants with 3–5 residues changed to alanine have no measurable differences from wild-type IL-23 except for binding of and signaling blockade by the 7B7 anti-IL-23 antibody. The M5 IL-23 mutant differs from wild-type by five alanine substitutions and represents the dominant energetic epitope of 7B7. M5 shows a dramatic decrease in binding to BMS-986010 (which contains the 7B7 Fab), yet maintains functional activity, binding to p40 and p19 specific reagents, and maintains biophysical properties similar to wild-type IL-23 (monomeric state, thermal stability, and secondary structural features).
Emulsion systems involving surfactants are mainly driven by the separation of the hydrophobic interactions of the aliphatic chains from the hydrophilic interactions of amphiphilic molecules in water. In this study, we report an emulsion system that does not include amphiphilic molecules but molecules with functional groups that are completely solvated in water. These functional groups give rise to molecular interactions including hydrogen bonding, pi stacking, and salt bridging and are segregated into a dispersion of droplets forming a water-in-water emulsion. This water-in-water emulsion consists of dispersing droplets of a water-solvated biocompatible liquid crystal--disodium cromoglycate (DSCG)--in a continuous aqueous solution containing specific classes of water-soluble polymers. Whereas aqueous solutions of polyols support the formation of emulsions of spherical droplets consisting of lyotropic liquid crystal DSCG with long-term stability (for at least 30 days), aqueous solutions of polyamides afford droplets of DSCG in the shape of prolate ellipsoids that are stable for only 2 days. The DSCG liquid crystal in spherical droplets assumes a radial configuration in which the optical axis of the liquid crystal aligns perpendicular to the surface of the droplets but assumes a tangential configuration in prolate ellipsoids in which the optical axis of the liquid crystal aligns parallel to the surface of the droplet. Other classes of water-soluble polymers including polyethers, polycations, and polyanions do not afford a stable emulsion of DSCG droplets. Both the occurrence and the stability of this unique emulsion system can be rationalized on the basis of the functional groups of the polymer. The different configurations of the liquid crystal (DSCG) droplets were also found to correlate with the strength of the hydrogen bonding that can be formed by the functional groups on the polymer.
This work reports a new biocompatible surfactant structure, of which the hydrophilic head group is composed of a folded, stable self-inclusion complex of a ferrocenyl substituted beta-cyclodextrin (betaCD). While multiple intra- or intermolecular complexes can exist for this amphiphile, the molecule folds into a unique intramolecular complex with well-defined conformation, in which part of the aliphatic chain and the ferrocene group are both included in the annular cavity of betaCD. Study of different isosteric covalent linkages indicates that this folded structure is stable against displacement by the presence of other small guest molecules. Furthermore, in contrast to ferrocene-CD conjugates that are without the aliphatic chain, the presence of small guest molecules in solution does not influence at all the induced circular dichroism signal of this amphiphile, indicating a sterically congested, but stable, folded conformation of the inclusion complex. This new amphiphile is surface active and, more importantly, does not denature the membrane protein bacteriorhodopsin. Finally, because this surfactant forms self-assembled aggregates, this work introduces a folded structure into soft matters formed by amphiphiles in water.
Here, we report a new class of highly chemoselective reactions between squarate derivatives and the amino acid cysteine or unprotected peptides with a N-terminus cysteine that proceed most efficiently in entirely aqueous solution at neutral pH. Kinetic and structural studies reveal that the presence of hydrogen bonding in water is primarily responsible for both the high yield and fast rate of the reaction.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.