John M. Franck

Quantitative cw Overhauser effect dynamic nuclear polarization for the analysis of local water dynamics.

Liquid state Overhauser effect Dynamic Nuclear Polarization (ODNP) has experienced a recent resurgence of interest. The ODNP technique described here relies on the double resonance of electron spin resonance (ESR) at the most common, i.e. X-band (∼10GHz), frequency and ¹H nuclear magnetic resonance (NMR) at ∼15 MHz. It requires only a standard continuous wave (cw) ESR spectrometer with an NMR probe inserted or built into an X-band cavity. We focus on reviewing a new and powerful manifestation of ODNP as a high frequency NMR relaxometry tool that probes dipolar cross relaxation between the electron spins and the ¹H nuclear spins at X-band frequencies. This technique selectively measures the translational mobility of water within a volume extending 0.5-1.5 nm outward from a nitroxide radical spin probe that is attached to a targeted site of a macromolecule. It allows one to study the dynamics of water that hydrates or permeates the surface or interior of proteins, polymers, and lipid membrane vesicles. We begin by reviewing the recent advances that have helped develop ODNP into a tool for mapping the dynamic landscape of hydration water with sub-nanometer locality. In order to bind this work coherently together and to place it in the context of the extensive body of research in the field of NMR relaxometry, we then rephrase the analytical model and extend the description of the ODNP-derived NMR signal enhancements. This extended model highlights several aspects of ODNP data analysis, including the importance of considering all possible effects of microwave sample heating, the need to consider the error associated with various relaxation rates, and the unique ability of ODNP to probe the electron-¹H cross-relaxation process, which is uniquely sensitive to fast (tens of ps) dynamical processes. By implementing the relevant corrections in a stepwise fashion, this paper draws a consensus result from previous ODNP procedures and then shows how such data can be further corrected to yield clear and reproducible saturation of the NMR hyperpolarization process. Finally, drawing on these results, we broadly survey the previous ODNP dynamics literature. We find that the vast number of published, empirical hydration dynamics data can be reproducibly classified into regimes of surface, interfacial, vs. buried water dynamics.

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.

Anomalously Rapid Hydration Water Diffusion Dynamics Near DNA Surfaces.

The emerging Overhauser effect dynamic nuclear polarization (ODNP) technique measures the translational mobility of water within the vicinity (5-15 Å) of preselected sites. The work presented here expands the capabilities of the ODNP technique and illuminates an important, previously unseen, property of the translational diffusion dynamics of water at the surface of DNA duplexes. We attach nitroxide radicals (i.e., spin labels) to multiple phosphate backbone positions of DNA duplexes, allowing ODNP to measure the hydration dynamics at select positions along the DNA surface. With a novel approach to ODNP analysis, we isolate the contributions of water molecules at these sites that undergo free translational diffusion from water molecules that either loosely bind to or exchange protons with the DNA. The results reveal that a significant population of water in a localized volume adjacent to the DNA surface exhibits fast, bulk-like characteristics and moves unusually rapidly compared to water found in similar probe volumes near protein and membrane surfaces. Control studies show that the observation of these characteristics are upheld even when the DNA duplex is tethered to streptavidin or the mobility of the nitroxides is altered. This implies that, as compared to protein or lipid surfaces, it is an intrinsic feature of the DNA duplex surface that it interacts only weakly with a significant fraction of the surface hydration water network. The displacement of this translationally mobile water is energetically less costly than that of more strongly bound water by up to several kBT and thus can lower the activation barrier for interactions involving the DNA surface.

Nonlinear Scaling of Surface Water Diffusion with Bulk Water Viscosity of Crowded Solutions

The translational hydration dynamics within 0.5-1.5 nm of the surface of a DPPC liposome, a model biomacromolecular surface, is analyzed by the recently developed Overhauser dynamic nuclear polarization (ODNP) technique. We find that dramatic changes to the bulk solvent cause only weak changes in the surface hydration dynamics. Specifically, both a >10-fold increase in bulk viscosity and the restriction of diffusion by confinement on a multiple nm length-scale change the local translational diffusion coefficient of the surface water surrounding the lipid bilayer by <2.5-fold. By contrast, previous ODNP studies have shown that changes to the biomacromolecular surface induced by folding, binding, or aggregation can cause local hydration dynamics to vary by factors of up to 30. We suggest that the surface topology and chemistry at the ≤1.5 nm scale, rather than the characteristics of the solvent, nearly exclusively determine the macromolecules surface hydration dynamics.

Specific ions modulate diffusion dynamics of hydration water on lipid membrane surfaces.

Effects of specific ions on the local translational diffusion of water near large hydrophilic lipid vesicle surfaces were measured by Overhauser dynamic nuclear polarization (ODNP). ODNP relies on an unpaired electron spin-containing probe located at molecular or surface sites to report on the dynamics of water protons within ∼10 Å from the spin probe, which give rise to spectral densities for electron–proton cross-relaxation processes in the 10 GHz regime. This pushes nuclear magnetic resonance relaxometry to more than an order of magnitude higher frequencies than conventionally feasible, permitting the measurement of water moving with picosecond to subnanosecond correlation times. Diffusion of water within ∼10 Å of, i.e., up to ∼3 water layers around the spin probes located on hydrophilic lipid vesicle surfaces is ∼5 times retarded compared to the bulk water translational diffusion. This directly reflects on the activation barrier for surface water diffusion, i.e., how tightly water is bound to the hydrophilic surface and surrounding waters. We find this value to be modulated by the presence of specific ions in solution, with its order following the known Hofmeister series. While a molecular description of how ions affect the hydration structure at the hydrophilic surface remains to be answered, the finding that Hofmeister ions directly modulate the surface water diffusivity implies that the strength of the hydrogen bond network of surface hydration water is directly modulated on hydrophilic surfaces.

Asymmetric Collapse in Biomimetic Complex Coacervates Revealed by Local Polymer and Water Dynamics

Complex coacervation is a phenomenon characterized by the association of oppositely charged polyelectrolytes into micrometer-scale liquid condensates. This process is the purported first step in the formation of underwater adhesives by sessile marine organisms, as well as the process harnessed for the formation of new synthetic and protein-based contemporary materials. Efforts to understand the physical nature of complex coacervates are important for developing robust adhesives, injectable materials, or novel drug delivery vehicles for biomedical applications; however, their internal fluidity necessitates the use of in situ characterization strategies of their local dynamic properties, capabilities not offered by conventional techniques such as X-ray scattering, microscopy, or bulk rheological measurements. Herein, we employ the novel magnetic resonance technique Overhauser dynamic nuclear polarization enhanced nuclear magnetic resonance (DNP), together with electron paramagnetic resonance (EPR) line shape analysis, to concurrently quantify local molecular and hydration dynamics, with species- and site-specificity. We observe striking differences in the structure and dynamics of the protein-based biomimetic complex coacervates from their synthetic analogues, which is an asymmetric collapse of the polyelectrolyte constituents. From this study we suggest charge heterogeneity within a given polyelectrolyte chain to be an important parameter by which the internal structure of complex coacervates may be tuned. Acquiring molecular-level insight to the internal structure and dynamics of dynamic polymer complexes in water through the in situ characterization of site- and species-specific local polymer and hydration dynamics should be a promising general approach that has not been widely employed for materials characterization.

Probing water density and dynamics in the chaperonin GroEL cavity

ATP-dependent binding of the chaperonin GroEL to its cofactor GroES forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution. It has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement, interactions with the cavity walls, and differences between the properties of cavity-confined and bulk water. However, experimental data regarding the cavity-confined water are lacking. Here, we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the Tyr71 → Cys GroES mutant obtained using two magnetic resonance techniques: electron-spin echo envelope modulation and Overhauser dynamic nuclear polarization. Residue 71 in GroES is fully exposed to bulk water in free GroES and to confined water within the cavity of the GroEL–GroES complex. Our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation, thus indicating that bulk water-exposed and cavity-confined GroES surface water share similar properties. Interestingly, the diffusion dynamics of water near the GroES surface are found to be unusually fast relative to other protein surfaces studied. The implications of these findings for chaperonin-assisted folding mechanisms are discussed.

Shimmed matching pulses: simultaneous control of rf and static gradients for inhomogeneity correction.

Portable NMR systems generally suffer from poor field homogeneity and are therefore used more commonly for imaging and relaxation measurements rather than for spectroscopy. In recent years, various approaches have been proposed to increase the sample volume that is usable for spectroscopy. These include approaches based on manual shimming and those based on clever combinations of modulated radio frequency and gradient fields. However, this volume remains small and, therefore, of limited utility. We present improved pulses designed to correct for inhomogeneous dispersion across wide ranges of frequency offsets without eliminating chemical shift or spatial encoding. This method, based on the adiabatic double passage, combines the relatively larger corrections available from spatially matched rf gradients [C. Meriles et al., J. Magn. Reson. 164, 177 (2003)]. with the adjustable corrections available from time-modulated static field gradients [D. Topgaard et al., Proc. Natl. Acad. Sci. U.S.A. 101, 17576 (2004)]. We explain the origins of these corrections with a theoretical model that simplifies and expedites the design of the pulse waveforms. We also present a generalized method for evaluating and comparing pulses designed for inhomogeneity correction. Experiments validate this method and support simulations that offer new possibilities for significantly enhanced performance in portable environments.

Least Squares Magnetic-Field Optimization for Portable Nuclear Magnetic Resonance Magnet Design

Single-sided and mobile nuclear magnetic resonance (NMR) sensors have the advantages of portability, low cost, and low power consumption compared to conventional high-field NMR and magnetic resonance imaging (MRI) systems. We present fast, flexible, and easy-to-implement target field algorithms for mobile NMR and MRI magnet design. The optimization finds a global optimum in a cost function that minimizes the error in the target magnetic field in the sense of least squares. When the technique is tested on a ring array of permanent-magnet elements, the solution matches the classical dipole Halbach solution. For a single-sided handheld NMR sensor, the algorithm yields a 640 G field homogeneous to 16 100 ppm across a 1.9 cm3 volume located 1.5 cm above the top of the magnets and homogeneous to 32 200 ppm over a 7.6 cm3 volume. This regime is adequate for MRI applications. We demonstrate that the homogeneous region can be continuously moved away from the sensor by rotating magnet rod elements, opening the way for NMR sensors with adjustable ldquosensitive volumes.rdquo

Active Cancellation – A Means to Zero Dead-Time Pulse EPR

The necessary resonator employed in pulse electron paramagnetic resonance (EPR) rings after the excitation pulse and creates a finite detector dead-time that ultimately prevents the detection of signal from fast relaxing spin systems, hindering the application of pulse EPR to room temperature measurements of interesting chemical or biological systems. We employ a recently available high bandwidth arbitrary waveform generator (AWG) to produce a cancellation pulse that precisely destructively interferes with the resonant cavity ring-down. We find that we can faithfully detect EPR signal at all times immediately after, as well as during, the excitation pulse. This is a proof of concept study showcasing the capability of AWG pulses to precisely cancel out the resonator ring-down, and allow for the detection of EPR signal during the pulse itself, as well as the dead-time of the resonator. However, the applicability of this approach to conventional EPR experiments is not immediate, as it hinges on either (1) the availability of low-noise microwave sources and amplifiers to produce the necessary power for pulse EPR experiment or (2) the availability of very high conversion factor micro coil resonators that allow for pulse EPR experiments at modest microwave power.