Hydration dynamics as an intrinsic ruler for refining protein structure at lipid membrane interfaces.

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.

α-Synuclein oligomers with broken helical conformation form lipoprotein nanoparticles.

α-Synuclein (αS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as "discs." Previously, it has been unclear whether αS also possesses this property. Using cryo-electron microscopy and light scattering, we found that αS can remodel phosphatidylglycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7-10-nm range) resemble those formed by apolipoproteins. The molar ratio of αS to lipid in nanoparticles is ∼1:20, and αS is oligomeric (including trimers and tetramers). Similar nanoparticles form when αS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by αS. Circular dichroism and four-pulse double electron electron resonance experiments revealed that in nanoparticles αS assumes a broken helical conformation distinct from the extended helical conformation adopted when αS is bound to intact vesicles or membrane tubules. We also observed αS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in a similar manner. αS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein.

Membrane curvature induction and tubulation are common features of synucleins and apolipoproteins.

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, ß-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.