Ravinath Kausik

Nature of Interactions between PEO-PPO-PEO Triblock Copolymers and Lipid Membranes: (II) Role of Hydration Dynamics Revealed by Dynamic Nuclear Polarization

Amphiphilic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymers, also known as poloxamers, have broad biomembrane activities. To illustrate the nature of these activities, (1)H Overhauser dynamic nuclear polarization NMR spectroscopy was employed to sensitively detect polymer-lipid membrane interactions through the modulation of local hydration dynamics in lipid membranes. Our study shows P188, the most hydrophilic poloxamer that is a known membrane sealant, weakly adsorbs on the membrane surface, yet effectively retards membrane hydration dynamics. Contrarily, P181, the most hydrophobic poloxamer that is a known membrane permeabilizer, initially embeds at lipid headgroups and enhances intrabilayer water diffusivity. Unprecedented resolution for differentiating weak surface adsorption versus translocation of polymers to membranes is obtained by probing local water diffusivity in lipid bilayer systems. Our results illustrate that the relative hydrophilic/hydrophobic ratio of the polymer dictates its functions. These findings gleaned from local hydration dynamics are well supported by a thermodynamics study presented in the accompanying paper (Wang, J.-Y.; Marks, J. M.; Lee, K. Y. C. Biomacromolecules, 2012, DOI: 10.1021/bm300847x).

Probing the hydration water diffusion of macromolecular surfaces and interfaces

We probe the translational dynamics of the hydration water surrounding the macromolecular surfaces of selected polyelectrolytes, lipid vesicles and intrinsically disordered proteins with site specificity in aqueous solutions. These measurements are made possible by the recent development of a new instrumental and methodological approach based on Overhauser dynamic nuclear polarization (DNP)-enhanced nuclear magnetic resonance (NMR) spectroscopy. This technique selectively amplifies 1H NMR signals of hydration water around a spin label that is attached to a molecular site of interest. The selective 1H NMR amplification within molecular length scales of a spin label is achieved by utilizing short-distance range (~r−3) magnetic dipolar interactions between the 1H spin of water and the electron spin of a nitroxide radical-based label. Key features include the fact that only minute quantities (<10 μl) and dilute (≥100 μM) sample concentrations are needed. There is no size limit on the macromolecule or molecular assembly to be analyzed. Hydration water with translational correlation times between 10 and 800 ps is measured within ~10 A distance of the spin label, encompassing the typical thickness of a hydration layer with three water molecules across. The hydration water moving within this time scale has significant implications, as this is what is modulated whenever macromolecules or molecular assemblies undergo interactions, binding or conformational changes. We demonstrate, with the examples of polymer complexation, protein aggregation and lipid–polymer interaction, that the measurements of interfacial hydration dynamics can sensitively and site specifically probe macromolecular interactions.

Ultrasensitive detection of interfacial water diffusion on lipid vesicle surfaces at molecular length scales.

Measurements of the interfacial diffusion coefficient of the surface hydration layer of lipid vesicles in dilute solutions are presented. This was made possible by the greatly enhanced sensitivity and unique contrast provided by the site-specific and selective Overhauser dynamic nuclear polarization of solvent molecules that approach nitroxide radical-based spin labels within <5-10 A. All experiments were carried out using minute microliter sample volumes of lipid vesicle solutions, using low spin label concentrations (<2 mol %) and under physiological conditions. This presents unprecedented sensitivity for analyzing interfacial solvent diffusion of macromolecules and their assemblies in solutions and highlights the feasibility of investigating precious samples. Interfacial diffusion on DOTAP (1,2-DiOleoyl-3-TrimethylAmmonium-Propane) and DPPC (1,2-DiPalmitoyl-sn-glycero-3-PhosphoCholine) surfaces are further analyzed as a function of temperature to determine the activation energy of their hydration layer dynamics. The temperature-dependent analysis across the phase transition of DPPC concludes that the hydration water with 100-200 ps dynamics displays Arrhenius behavior and does not undergo a phase transition unlike the lipid chains. We also discuss the advantages of determining the activation energy of diffusion as a general approach to comparing interfacial diffusivity on surfaces that have vastly different charge topologies and, thus, may display different distances of closest approach between the spin label placed at the surface and the protons of hydration water. The further development and application of this technique is expected to facilitate the study of membrane dynamics and their phase behavior, including the formation of lipid rafts, with lipid-specific resolution.

Dynamic nuclear polarization enhanced nuclear magnetic resonance and electron spin resonance studies of hydration and local water dynamics in micelle and vesicle assemblies.

We present a unique analysis tool for the selective detection of local water inside soft molecular assemblies (hydrophobic cores, vesicular bilayers, and micellar structures) suspended in bulk water. Through the use of dynamic nuclear polarization (DNP), the (1)H NMR signal of water is amplified, as it interacts with stable radicals that possess approximately 658 times higher spin polarization. We utilized stable nitroxide radicals covalently attached along the hydrophobic tail of stearic acid molecules that incorporate themselves into surfactant-based micelle or vesicle structures. Here, we present a study of local water content and fluid viscosity inside oleate micelles and vesicles and Triton X-100 micelles to serve as model systems for soft molecular assemblies. This approach is unique because the amplification of the NMR signal is performed in bulk solution and under ambient conditions with site-specific spin labels that only detect the water that is directly interacting with the localized spin labels. Continuous wave (cw) electron spin resonance (ESR) analysis provides rotational dynamics of the spin-labeled molecular chain segments and local polarity parameters that can be related to hydration properties, whereas we show that DNP-enhanced (1)H NMR analysis of fluid samples directly provides translational water dynamics and permeability of the local environment probed by the spin label. Our technique therefore has the potential to become a powerful analysis tool, complementary to cw ESR, to study hydration characteristics of surfactant assemblies, lipid bilayers, or protein aggregates, where water dynamics is a key parameter of their structure and function. In this study, we find that there is significant penetration of water inside the oleate micelles with a higher average local water viscosity (approximately 1.8 cP) than in bulk water, and Triton X-100 micelles and oleate vesicle bilayers mostly exclude water while allowing for considerable surfactant chain motion and measurable water permeation through the soft structure.

An ultrasensitive tool exploiting hydration dynamics to decipher weak lipid membrane–polymer interactions

We introduce a newly developed tool, (1)H Overhauser Dynamic Nuclear Polarization (ODNP), to sensitively explore weak macromolecular interactions by site-specifically probing the modulation of the translational dynamics of hydration water at the interaction interface, in the full presence of bulk water. Here, ODNP is employed on an illustrative example of a membrane-active triblock copolymer, poloxamer 188 (P188), which is known to restore the integrity of structurally compromised cell membranes. We observe a distinct change in the translational dynamics of the hydration layer interacting with the lipid membrane surface and the bilayer-interior as P188 is added to a solution of lipid vesicles, but no measurable changes in the dynamics or structure of the lipid membranes. This study shows that hydration water is an integral constituent of a lipid membrane system, and demonstrates for the first time that the modulation of its translational diffusivity can sensitively report on weak polymer-membrane interactions, as well as mediate essential lipid membrane functions. ODNP holds much promise as a unique tool to unravel molecular interactions at interfaces even in the presence of bulk water under ambient conditions.

Confinement effect of chain dynamics in micrometer thick layers of a polymer melt below the critical molecular weight

Polymer melts confined in micrometer thick layers were examined with the aid of field-cycling NMR relaxometry. It is shown that chain dynamics under such moderate confinement conditions are perceptibly different from those observed in the bulk material. This is considered to be a consequence of the corset effect, which predicts a crossover between Rouse and reptationlike dynamics for molecular weights below the critical value at confinement length scales much larger than 10RF, where RF is the Flory radius of the bulk polymer coil [Fatkullin et al., New J. Phys. 6, 46 (2004)]. For the polymer species studied, a perfluoropolyether with a molecular weight of 11 000, the Flory radius is of the order 10 nm, so that the experiment refers to the far end of the predicted crossover region from confined to bulk chain dynamics. Remarkably the confinement effect is shown to reach polymer-wall distances of the order 100 Flory radii.

Cooperative polymer dynamics under nanoscopic pore confinements probed by field-cycling NMR relaxometry

Reptational dynamics of bulk polymer chains on a time scale between the Rouse mode relaxation time and the so-called disengagement time is not compatible with the basic thermodynamic law of fluctuations of the number of segments in a given volume. On the other hand, experimental field-cycling NMR relaxometry data of perfluoropolyether melts confined in Vycor, a porous silica glass of nominal pore dimension of 4 nm, closely display the predicted signatures for the molecular weight and frequency dependences of the spin-lattice relaxation time in this particular limit, namely T1 proportional M-1/2nu1/2. It is shown that this contradiction is an apparent one. In this paper a formalism is developed suggesting cooperative chain dynamics under nanoscopic pore confinements. The result is a cooperative reptational displacement phenomenon reducing the root-mean-squared displacement rate correspondingly but showing the same characteristic dependences as the ordinary reptation model. The tube diameter effective for cooperative reptation is estimated on this basis for the sample system under consideration and is found to be of the same order of magnitude as the nominal pore diameter of Vycor.

Cholesterol enhances surface water diffusion of phospholipid bilayers

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.

Sensitivity and resolution of two-dimensional NMR diffusion-relaxation measurements.

The performance of 2D NMR diffusion-relaxation measurements for fluid typing applications is analyzed. In particular, we delineate the region in the diffusion - relaxation plane that can be determined with a given gradient strength and homogeneity, and compare the performance of the single and double echo encoding with the stimulated echo diffusion encoding. We show that the diffusion editing based approach is able to determine the diffusion coefficient only if the relaxation time T2 exceeds a cutoff value T2,cutoff, that scales like T2,cutoff∝g(-2/3)D(-1/3). For stimulated echo encoding, the optimal diffusion encoding times (Td and δ), that provide the best diffusion sensitivity, rely only on the T1/T2 ratios and not on the diffusion coefficients of the fluids or the applied gradient strengths. Irrespective of T1, for high enough gradients (i.e. when γ(2)g(2)DT2(3)>10(2)), the Hahn echo based encoding is superior to encoding based on the stimulated echo. For weaker gradients, the stimulated echo is superior only if the T1/T2 ratio is much larger than 1. For single component systems, the diffusion sensitivity is not adversely impacted by the uniformity of the gradients and the diffusion distributions can be well measured. The presence of non-uniform gradients can affect the determination of the diffusion distributions when you have two fluids of comparable T2. In such situations the effective single component diffusion coefficient is always closer to the geometric mean diffusion coefficient of the two fluids.

Local Hydration Dynamics in Soft Matter by Dynamic Nuclear Polarization of 1H‐Water

We present a unique approach for measuring the local diffusion coefficient of water inside or at the surface of soft matter systems, including lipid bilayer membranes, proteins, or protein assemblies. This is made possible by the use of dynamic nuclear polarization (DNP) of 1H NMR signal of water via nitroxide based spin labels which are localized onto specific membrane or protein sites through covalent linkage. The DNP‐induced 1H NMR signal enhancement critically depends on the diffusion coefficient of the solvent within approximately 10 A distance of the spin labeled site. We discuss the usefulness of 1H NMR in studying local water dynamics and accessibility in combination with electron spin spectroscopy and compare the results to the complementary technique of field cycling relaxometry (FC). Most importantly, the DNP approach allows us to work with sample volumes as small as 3–4 μL at spin label concentrations nearing 100 μM giving ∼2 orders of magnitude higher sensitivity compared with field cycling a...