Mapping invisible epitopes by NMR spectroscopy

Abstract

Defining discontinuous antigenic epitopes remains a substantial challenge, as exemplified by the case of lipid transfer polyproteins, which are common pollen allergens. Hydrogen/deuterium exchange monitored by NMR can be used to map epitopes onto folded protein surfaces, but only if the complex rapidly dissociates. Modifying the standard NMR-exchange measurement to detect substoichiometric complexes overcomes this time scale limitation and provides new insights into recognition of lipid transfer polyprotein by antibodies. In the future, this new and exciting development should see broad application to a range of tight macromolecular interactions.

Figure 1.

The HDXMEM technique and its potential applications to slowly dissociating macromolecular complexes. When a protein of interest (POI) (pink) associates very tightly with a large binding partner or assembly, it is unfeasible to detect the bound-state POI (gray) by traditional NMR methods. HDXMEM relies on protection of the binding surface in the NMR-invisible state under subsaturating conditions, where the unbound POI can be detected with a “memory” of the bound state. HDXMEM may advance detection and description of high-affinity, slowly dissociating complexes, such as protein-protein and protein-RNA interactions, the formation of amyloid fibrils, and dense-dilute phase equilibria.