Hydration Dynamics as a Molecular Ruler for Refining Protein Structure at Lipid Membrane Interfaces

Abstract

Although the 3D protein structure provides the molecular basis for unraveling its unique function, the structural characterization of protein at lipid membrane interfaces is experimentally challenging under physiological conditions. Here, we demonstrate the feasibility of using the long-range and distinct gradient in hydration dynamics along the bilayer normal as a molecular ruler to determine the secondary structure, orientation, and immersion depth of a membrane-associating protein. It was made possible through the quantification of translational diffusion of loosely bound hydration water at the specific sites of protein and lipid bilayer using our newly developed method, Overhauser Dynamic Nuclear Polarization (ODNP)-1H NMR spectroscopy. This study is demonstrated on α-synuclein (αS), a protein associated with Parkinson's disease, free in solution vs. bound on an overall negatively charged phospholipid vesicle. ODNP exhibits that the membrane-bound αS forms an α-helix from residue-76 to 90, where the center of the α-helix is positioned ∼1 below the lipid phosphate. Our data extends the current understanding by showing that residues 90 to 96 of bound αS form a transition segment that links the α-helical domain and the C-terminus with a larger turn than an idealized α-helix, and that the unstructured C-terminus gradually threads outward through a distinct hydration dynamics gradient spanning 10-20 between the water-membrane interface and bulk solvent. Remarkably, ODNP enables to resolve the structural nature of protein functional domains residing 10-20 above the lipid phosphate, which it is difficult to access experimentally. This study debuts the hydration dynamics gradient at protein-lipid interfaces as a high-resolution molecular ruler for probing the structure of membrane proteins and peptides, whose membrane-bound conformation, location and hydration signature are critical to understanding their biological functions.