Knowing the topology and location of protein segments at water-membrane interfaces is critical for rationalizing their functions, but their characterization is challenging under physiological conditions. Here, we debut a unique spectroscopic approach by using the hydration dynamics gradient found across the phospholipid bilayer as an intrinsic ruler for determining the topology, immersion depth, and orientation of protein segments in lipid membranes, particularly at water-membrane interfaces. This is achieved through the site-specific quantification of translational diffusion of hydration water using an emerging tool, (1)H Overhauser dynamic nuclear polarization (ODNP)-enhanced NMR relaxometry. ODNP confirms that the membrane-bound region of α-synuclein (αS), an amyloid protein known to insert an amphipathic α-helix into negatively charged phospholipid membranes, forms an extended α-helix parallel to the membrane surface. We extend the current knowledge by showing that residues 90-96 of bound αS, which is a transition segment that links the α-helix and the C terminus, adopt a larger loop than an idealized α-helix. The unstructured C terminus gradually threads through the surface hydration layers of lipid membranes, with the beginning portion residing within 5-15 Å above the phosphate level, and only the very end of C terminus surveying bulk water. Remarkably, the intrinsic hydration dynamics gradient along the bilayer normal extends to 20-30 Å above the phosphate level, as demonstrated with a peripheral membrane protein, annexin B12. ODNP offers the opportunity to reveal previously unresolvable structure and location of protein segments well above the lipid phosphate, whose structure and dynamics critically contribute to the understanding of functional versatility of membrane proteins.

Antibodies hold significant potential for inhibiting toxic protein aggregation associated with conformational disorders such as Alzheimer's and Huntington's diseases. However, near-stoichiometric antibody concentrations are typically required to completely inhibit protein aggregation. We posited that the molecular interactions mediating amyloid fibril formation could be harnessed to generate antibodies with potent antiaggregation. Here we report that grafting small amyloidogenic peptides (6-10 residues) into the complementarity-determining regions of a single-domain (V(H)) antibody yields potent domain antibody inhibitors of amyloid formation. Grafted AMyloid-Motif AntiBODIES (gammabodies) presenting hydrophobic peptides from Aβ (Alzheimer's disease), α-Synuclein (Parkinson's disease), and islet amyloid polypeptide (type 2 diabetes) inhibit fibril assembly of each corresponding polypeptide at low substoichiometric concentrations (1:10 gammabody:monomer molar ratio). In contrast, sequence- and conformation-specific antibodies that were obtained via immunization are unable to prevent fibrillization at the same substoichiometric concentrations. Gammabodies prevent amyloid formation by converting monomers and/or fibrillar intermediates into small complexes that are unstructured and benign. We expect that our antibody design approach--which eliminates the need for immunization or screening to identify sequence-specific domain antibody inhibitors--can be readily extended to generate potent aggregation inhibitors of other amyloidogenic polypeptides linked to human disease.