Site-Specific Hydration Dynamics in the Nonpolar Core of a Molten Globule by Dynamic Nuclear Polarization of Water

Proc. Natl. Acad. Sci. U.S.A.

Varkey

Ambroso

et al. 2013

Self Cite

Knowing the topology and location of protein segments at watermembrane 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 sitespecific 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.electron paramagnetic resonance | site-directed spin labeling | protein-lipid interaction | liposomes | Parkinson disease

Section: Topology and Immersion Depth Of α-Synuclein At The Water-memsupporting

confidence: 60%

Section: Approach To Quantify Local Hydration Dynamics At Biomolecularmentioning

confidence: 96%

Section: Significancementioning

confidence: 99%

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

Proc. Natl. Acad. Sci. U.S.A.

Varkey

Ambroso

et al. 2013

Self Cite

“…In order to compare water diffusivity in different local environments, we introduce the retardation factor, τ /τ bulk , which is the ratio of the τ value of hydration water to that of bulk water, τ bulk . The retardation factor is typically 2-5 for hydration water on water-exposed surfaces of protein or lipid membrane, 6,57,68,75,76 whereas it is around 5-11 in the bilayer interior of lipid assemblies. 72,77,78 However, the relatively modest retardation factor for water within the lipid bilayer only reports on the relatively fast diffusion dynamics of the highly sparse water molecules across the bilayer, but does not provide any information about water content.…”

Section: −1mentioning

confidence: 99%

Cholesterol enhances surface water diffusion of phospholipid bilayers

The Journal of Chemical Physics

Olijve

Kausik

et al. 2014

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Elucidating the physical effect of cholesterol (Chol) on biological membranes is necessary towards rationalizing their structural and functional role in cell membranes. One of the debated questions is the role of hydration water in Chol-embedding lipid membranes, for which only little direct experimental data are available. Here, we study the hydration dynamics in a series of Chol-rich and depleted bilayer systems using an approach termed 1 H Overhauser dynamic nuclear polarization (ODNP) NMR relaxometry that enables the sensitive and selective determination of water diffusion within 5-10 Å of a nitroxide-based spin label, positioned off the surface of the polar headgroups or within the nonpolar core of lipid membranes. The Chol-rich membrane systems were prepared from mixtures of Chol, dipalmitoyl phosphatidylcholine and/or dioctadecyl phosphatidylcholine lipid that are known to form liquid-ordered, raft-like, domains. Our data reveal that the translational diffusion of local water on the surface and within the hydrocarbon volume of the bilayer is significantly altered, but in opposite directions: accelerated on the membrane surface and dramatically slowed in the bilayer interior with increasing Chol content. Electron paramagnetic resonance (EPR) lineshape analysis shows looser packing of lipid headgroups and concurrently tighter packing in the bilayer core with increasing Chol content, with the effects peaking at lipid compositions reported to form lipid rafts. The complementary capability of ODNP and EPR to site-specifically probe the hydration dynamics and lipid ordering in lipid membrane systems extends the current understanding of how Chol may regulate biological processes. One possible role of Chol is the facilitation of interactions between biological constituents and the lipid membrane through the weakening or disruption of strong hydrogen-bond networks of the surface hydration layers that otherwise exert stronger repulsive forces, as reflected in faster surface water diffusivity. Another is the concurrent tightening of lipid packing that reduces passive, possibly unwanted, diffusion of ions and water across the bilayer.

“…We have previously established a broadly applicable spectroscopic method, Overhauser dynamic nuclear polarizationenhanced NMR relaxometry (ODNP) (16,17), to probe changes in translational diffusivity of local water within 1 nm of nitroxide radical-based electron spin labels tethered to specific protein residues, and successfully reported on the study of protein folding, protein aggregation, and conformational changes of globular and membrane protein segments (18)(19)(20)(21). ODNP has revealed increased heterogeneity, i.e., dispersion, in local water diffusivity on the surface of a folded protein, compared with its unfolded counterpart or folding intermediate (20).…”

mentioning

confidence: 99%

Protein structural and surface water rearrangement constitute major events in the earliest aggregation stages of tau

Pavlova

Proc. Natl. Acad. Sci. U.S.A.

Kinnebrew

et al. 2015

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Protein aggregation plays a critical role in the pathogenesis of neurodegenerative diseases, and the mechanism of its progression is poorly understood. Here, we examine the structural and dynamic characteristics of transiently evolving protein aggregates under ambient conditions by directly probing protein surface water diffusivity, local protein segment dynamics, and interprotein packing as a function of aggregation time, along the third repeat domain and C terminus of Δtau187 spanning residues 255-441 of the longest isoform of human tau. These measurements were achieved with a set of highly sensitive magnetic resonance tools that rely on sitespecific electron spin labeling of Δtau187. Within minutes of initiated aggregation, the majority of Δtau187 that is initially homogeneously hydrated undergoes structural transformations to form partially structured aggregation intermediates. This is reflected in the dispersion of surface water dynamics that is distinct around the third repeat domain, found to be embedded in an intertau interface, from that of the solvent-exposed C terminus. Over the course of hours and in a rate-limiting process, a majority of these aggregation intermediates proceed to convert into stable β-sheet structured species and maintain their stacking order without exchanging their subunits. The population of β-sheet structured species is >5% within 5 min of aggregation and gradually grows to 50-70% within the early stages of fibril formation, while they mostly anneal block-wisely to form elongated fibrils. Our findings suggest that the formation of dynamic aggregation intermediates constitutes a major event occurring in the earliest stages of tau aggregation that precedes, and likely facilitates, fibril formation and growth.biological water | soluble oligomers | amyloid formation | site-directed spin labeling | Alzheimer's disease