Alexandria Guinness - Academia.edu (original) (raw)

Papers by Alexandria Guinness

arXiv (Cornell University), Jun 10, 2023

This contribution demonstrates a two dimensional deuterium NMR methodology for discriminating bet... more This contribution demonstrates a two dimensional deuterium NMR methodology for discriminating between D2O populations whose properties differ as a result of being confined inside nanoscale volumes. Importantly, for reverse micelles (a proof-of-principal system), as the lengthscale of the confinement is changed from several nanometers down to less than a nanometer, the position of the signal peak migrates through the 2D spectrum, following a distinctive trend. This trend most typically involves relatively gentle linear change in the order of magnitude of the NMR relaxation time for water confined on the scale of several nanometers, followed by a region of dramatic negative curvature (of relaxation time vs. chemical shift) for water confined to lengthscales smaller than 1-2 nanometers. Interestingly, the qualitative shape of this trend can change with different choices of surfactants, i.e., a different choice of the chemistry at the edges of the confining environment. An important facet of this research was to demonstrate the relatively wide applicability of these techniques by showing that both: (1) Standard modern NMR instrumentation is capable of deploying an automated measurement, even though the choice of a deuterium nucleus is non standard and frequently requires companion proton spectra in order to reference the chemical shifts; and (2) well established (though underutilized) modern signal processing techniques can generate the resulting signal even though it involves the somewhat unusual combination of chemical shifts along one dimension and a distribution of relaxation times along another dimension. In addition to demonstrating that this technique can be deployed across many samples of interest, detailed facts pertaining to the broadening or shifting of resulting signals upon inclusion of various guests molecules are also discussed.

Journal of Chemical Physics, Nov 7, 2022

Over recent decades, the value of conducting experiments at lower frequencies and in inhomogeneou... more Over recent decades, the value of conducting experiments at lower frequencies and in inhomogeneous and/or time-variable fields has grown. For example, an interest in the nanoscale heterogeneities of hydration dynamics demands increasingly sophisticated and automated measurements deploying Overhauser Dynamic Nuclear Polarization (ODNP) at low field. The development of these methods poses various challenges that drove us to develop a standardized alternative to the traditional schema for acquiring and analyzing coherence pathway information employed by the overwhelming majority of contemporary Nuclear Magnetic Resonance (NMR) research. Specifically, on well-tested, stable NMR systems running well-tested pulse sequences in highly optimized, homogeneous magnetic fields, traditional hardware and software quickly isolate a meaningful subset of data by averaging and discarding between 3/4 and 127/128 of the digitized data. In contrast, spurred by recent advances in the capabilities of open-source libraries, the domain colored coherence transfer (DCCT) schema implemented here builds on the long-extant concept of Fourier transformation along the pulse phase cycle dimension to enable data visualization that more fully reflects the rich physics underlying these NMR experiments. In addition to discussing the outline and implementation of the general DCCT schema and associated plotting methods, this manuscript presents a collection of algorithms that provide robust phasing, avoidance of baseline distortion, and the ability to realize relatively weak signals amidst background noise through signal-averaged correlation alignment. The methods for visualizing the raw data, together with the processing routines whose development they guide should apply directly to or extend easily to other techniques facing similar challenges.

arXiv (Cornell University), Jul 6, 2023

Proteins involved in signaling pathways represent an interesting target for experimental analysis... more Proteins involved in signaling pathways represent an interesting target for experimental analysis by Overhauser Dynamic Nuclear Polarization (ODNP) (Overhauser Dynamic Nuclear Polarization), which determines the translational mobility at the surface of proteins. They also represent a challenge, since the hydration dynamics at all sites remains relatively rapid, requiring sensitive measurements capable of drawing finer distinctions. Targeting the protein K-Ras, we find ODNP cross-relaxivity values that appear consistent within similar regions of 3D space, regardless of the specific residue where the spin probe used to select the location has been attached. The similar dynamics observed from nearby residues indicate a persistence/uniformity of the translational dynamics of water on the nanometer scale. This results makes sense, since it essentially means that the dynamics of water remains consistent over a lengthscale (a nanometer) over which liquid water exhibits structural persistence (i.e. its correlation length). This opens up the possibility of strategically and comprehensively mapping out the hydration layer in aqueous solution and identifying regions that contribute significantly to the free energy of binding interactions-for example, slow water that might contribute significant entropy, or regions with strongly temperature-dependent water mobility that might contribute significant enthalpy.

Journal of Chemical Physics, Sep 13, 2022

Liquid state Overhauser Effect Dynamic Nuclear Polarization (ODNP) has experienced a recent resur... more Liquid state Overhauser Effect Dynamic Nuclear Polarization (ODNP) has experienced a recent resurgence of interest. In particular, a new manifestation of the ODNP measurement [1] measures the translational mobility of water within 5-10Å of an ESR-active spin probe (i.e. the local translational diffusivityD local near an electron spin resonance active molecule). Such spin probes, typically stable nitroxide radicals, have been attached to the surface or interior of macromolecules, including proteins [2, 3], polymers [4], and membrane vesicles [5]. Despite the unique specificity of this measurement, it requires only a standard X-band (∼10 GHz) continuous wave (cw) electron spin resonance (ESR) spectrometer, coupled with a standard nuclear magnetic resonance (NMR) spectrometer. Here, we present a set of developments and corrections that allow us to improve the accuracy of quantitative ODNP and apply it to samples more than two orders of magnitude lower than were previously feasible. An existing model for ODNP signal enhancements [6-9] accurately predicts the ODNP enhancements for water that contains high (≥ 10 mM) concentrations of spin probes, whether they be freely dissolved in solution [1, 6, 10] or covalently tethered to slowly tumbling macromolecular systems [1, 4]. This model yields a parameter called the coupling factor, ξ, which gives the efficiency of the ODNP polarization transfer in the presence of the spin label, and which depends only on the relative motion of the water molecules and the spin label. Measurements of the ODNP enhancements and relaxation times can extract the parameter ξ, allowing one to read out the local translational dynamics of the water near the spin probe. However, recent literature yields conflicting results for basic ODNP measurements of small spin probes dissolved in water [1, 6, 10, 11] and a closer inspection-especially at low concentrations of spin probes-reveals unexpected results that imply the breakdown of the existing model as a result of microwave-induced sample heating. Specifically, while the conventional model predicts that the enhancements should converge asymptotically to a maximum value, Emax, at high microwave powers, the enhancements instead continue to increase linearly. In part due to this breakdown of the model, the concentration regime below ∼100 µM was previously quite infeasible for quantitative Overhauser DNP studies. The technique presented here feasibly quantifies the ODNP coupling factor at lower concentrations by separately determining the two fundamental relaxivities involved in ODNP: the local crossrelaxivity, kσ, and the local self-relaxivity, kρ, whose ratio gives the coupling factor, ξ = kσ/kρ. These relaxivities determine the concentration-dependent relaxation rates for the cross relaxation from the electrons to the protons, and for the self-relaxation from the protons near the spin probe to the bath (i.e. "lattice"), respectively. Enhancement vs. power (E(p)) curves acquired on cw ODNP instrumentation can quantify the cross-relaxivity (kσ) for concentrations as low as tens of micromolar. Furthermore, such data can include a correction for the microwave heating effects previously mentioned. Independent measurements can provide accurate values for the self-relaxivity (kρ) that are not affected by microwave heating, and which will have even further improved accuracy when obtained from samples of larger volume or higher concentration. The more accurate value for the coupling factor, ξ, that results from this new technique more reliably quantifies the local translational diffusivity, D local , near the spin probe and opens up the novel possibility of analyzing lower sample concentrations of ≤ 100 µM that are critical for biomolecular studies. To demonstrate these improvements and compare to recent results, we repeat careful measurements of the coupling factor (ξ) between a small nitroxide probe (4-hydroxy-TEMPO) and otherwise unperturbed bulk water, at both high and low spin probe concentrations. At high concentrations, we measure a significantly higher extrapolated enhancement, Emax, than was previously measured or predicted by solely cw ODNP-based work [6]. At all concentrations, for the first time, the data measured by the cw ODNP instrumentation shown here agrees with the coupling factor values of 0.36 [1], 0.33-0.35 [12], or 0.33 [10, 11] that others have reported based on ODNP measurements augmented by FCR experiments and pulsed ESR experiments, or the value of 0.30 predicted by molecular dynamics simulations [13]. On the one hand, this observation resolves the debate revolving around the absolute value of the coupling factor between water and freely dissolved spin probes, which is an important reference value for the study of hydration water in biological and other macromolecular systems. Our data conclusively supports a values of 0.33 [10, 11] rather than 0.22 [1, 6]. On the other hand, contrary to conclusions drawn in previous literature [11, 14], this data implies that solely cw ODNP methods can provide quantitative and accurate coupling factors, and thus derive accurate hydration dynamics information. This is fortuitous; FCR and pulsed ESR tools will continue to present powerful and complementary capabilities, while the implementation of quantitative ODNP measurements on widely available and easy to use cw ODNP instrumentation has distinctly practical benefits for the end user.

Over recent decades, motivated either by practicality or the need to tap into new types of measur... more Over recent decades, motivated either by practicality or the need to tap into new types of measurements, the science of Magnetic Resonance has expanded into more adverse conditions: deliberately chosen lower frequencies, inhomogeneous fields, and/or time-variable fields. In particular, Overhauser Dynamic Nuclear Polarization (ODNP) presents a case study that challenges previous expectations and offers an interesting test-bed for further developments. For example, an interest in the nanoscale heterogeneities of hydration dynamics demand increasingly sophisticated and automated measurements deploying ODNP on a modular, open source instrument operating at 15 MHz. ODNP demands the acquisition and automated processing of large quantities of one dimensional NMR spectra, which can present various problems: in particular, unambiguous identification of signal in newly configured instruments presents a practical challenge, while field drift tends to remain an issue even in fully configured in...

arXiv (Cornell University), Jun 10, 2023

This contribution demonstrates a two dimensional deuterium NMR methodology for discriminating bet... more This contribution demonstrates a two dimensional deuterium NMR methodology for discriminating between D2O populations whose properties differ as a result of being confined inside nanoscale volumes. Importantly, for reverse micelles (a proof-of-principal system), as the lengthscale of the confinement is changed from several nanometers down to less than a nanometer, the position of the signal peak migrates through the 2D spectrum, following a distinctive trend. This trend most typically involves relatively gentle linear change in the order of magnitude of the NMR relaxation time for water confined on the scale of several nanometers, followed by a region of dramatic negative curvature (of relaxation time vs. chemical shift) for water confined to lengthscales smaller than 1-2 nanometers. Interestingly, the qualitative shape of this trend can change with different choices of surfactants, i.e., a different choice of the chemistry at the edges of the confining environment. An important facet of this research was to demonstrate the relatively wide applicability of these techniques by showing that both: (1) Standard modern NMR instrumentation is capable of deploying an automated measurement, even though the choice of a deuterium nucleus is non standard and frequently requires companion proton spectra in order to reference the chemical shifts; and (2) well established (though underutilized) modern signal processing techniques can generate the resulting signal even though it involves the somewhat unusual combination of chemical shifts along one dimension and a distribution of relaxation times along another dimension. In addition to demonstrating that this technique can be deployed across many samples of interest, detailed facts pertaining to the broadening or shifting of resulting signals upon inclusion of various guests molecules are also discussed.

Journal of Chemical Physics, Nov 7, 2022

Over recent decades, the value of conducting experiments at lower frequencies and in inhomogeneou... more Over recent decades, the value of conducting experiments at lower frequencies and in inhomogeneous and/or time-variable fields has grown. For example, an interest in the nanoscale heterogeneities of hydration dynamics demands increasingly sophisticated and automated measurements deploying Overhauser Dynamic Nuclear Polarization (ODNP) at low field. The development of these methods poses various challenges that drove us to develop a standardized alternative to the traditional schema for acquiring and analyzing coherence pathway information employed by the overwhelming majority of contemporary Nuclear Magnetic Resonance (NMR) research. Specifically, on well-tested, stable NMR systems running well-tested pulse sequences in highly optimized, homogeneous magnetic fields, traditional hardware and software quickly isolate a meaningful subset of data by averaging and discarding between 3/4 and 127/128 of the digitized data. In contrast, spurred by recent advances in the capabilities of open-source libraries, the domain colored coherence transfer (DCCT) schema implemented here builds on the long-extant concept of Fourier transformation along the pulse phase cycle dimension to enable data visualization that more fully reflects the rich physics underlying these NMR experiments. In addition to discussing the outline and implementation of the general DCCT schema and associated plotting methods, this manuscript presents a collection of algorithms that provide robust phasing, avoidance of baseline distortion, and the ability to realize relatively weak signals amidst background noise through signal-averaged correlation alignment. The methods for visualizing the raw data, together with the processing routines whose development they guide should apply directly to or extend easily to other techniques facing similar challenges.

arXiv (Cornell University), Jul 6, 2023

Proteins involved in signaling pathways represent an interesting target for experimental analysis... more Proteins involved in signaling pathways represent an interesting target for experimental analysis by Overhauser Dynamic Nuclear Polarization (ODNP) (Overhauser Dynamic Nuclear Polarization), which determines the translational mobility at the surface of proteins. They also represent a challenge, since the hydration dynamics at all sites remains relatively rapid, requiring sensitive measurements capable of drawing finer distinctions. Targeting the protein K-Ras, we find ODNP cross-relaxivity values that appear consistent within similar regions of 3D space, regardless of the specific residue where the spin probe used to select the location has been attached. The similar dynamics observed from nearby residues indicate a persistence/uniformity of the translational dynamics of water on the nanometer scale. This results makes sense, since it essentially means that the dynamics of water remains consistent over a lengthscale (a nanometer) over which liquid water exhibits structural persistence (i.e. its correlation length). This opens up the possibility of strategically and comprehensively mapping out the hydration layer in aqueous solution and identifying regions that contribute significantly to the free energy of binding interactions-for example, slow water that might contribute significant entropy, or regions with strongly temperature-dependent water mobility that might contribute significant enthalpy.

Journal of Chemical Physics, Sep 13, 2022

Liquid state Overhauser Effect Dynamic Nuclear Polarization (ODNP) has experienced a recent resur... more Liquid state Overhauser Effect Dynamic Nuclear Polarization (ODNP) has experienced a recent resurgence of interest. In particular, a new manifestation of the ODNP measurement [1] measures the translational mobility of water within 5-10Å of an ESR-active spin probe (i.e. the local translational diffusivityD local near an electron spin resonance active molecule). Such spin probes, typically stable nitroxide radicals, have been attached to the surface or interior of macromolecules, including proteins [2, 3], polymers [4], and membrane vesicles [5]. Despite the unique specificity of this measurement, it requires only a standard X-band (∼10 GHz) continuous wave (cw) electron spin resonance (ESR) spectrometer, coupled with a standard nuclear magnetic resonance (NMR) spectrometer. Here, we present a set of developments and corrections that allow us to improve the accuracy of quantitative ODNP and apply it to samples more than two orders of magnitude lower than were previously feasible. An existing model for ODNP signal enhancements [6-9] accurately predicts the ODNP enhancements for water that contains high (≥ 10 mM) concentrations of spin probes, whether they be freely dissolved in solution [1, 6, 10] or covalently tethered to slowly tumbling macromolecular systems [1, 4]. This model yields a parameter called the coupling factor, ξ, which gives the efficiency of the ODNP polarization transfer in the presence of the spin label, and which depends only on the relative motion of the water molecules and the spin label. Measurements of the ODNP enhancements and relaxation times can extract the parameter ξ, allowing one to read out the local translational dynamics of the water near the spin probe. However, recent literature yields conflicting results for basic ODNP measurements of small spin probes dissolved in water [1, 6, 10, 11] and a closer inspection-especially at low concentrations of spin probes-reveals unexpected results that imply the breakdown of the existing model as a result of microwave-induced sample heating. Specifically, while the conventional model predicts that the enhancements should converge asymptotically to a maximum value, Emax, at high microwave powers, the enhancements instead continue to increase linearly. In part due to this breakdown of the model, the concentration regime below ∼100 µM was previously quite infeasible for quantitative Overhauser DNP studies. The technique presented here feasibly quantifies the ODNP coupling factor at lower concentrations by separately determining the two fundamental relaxivities involved in ODNP: the local crossrelaxivity, kσ, and the local self-relaxivity, kρ, whose ratio gives the coupling factor, ξ = kσ/kρ. These relaxivities determine the concentration-dependent relaxation rates for the cross relaxation from the electrons to the protons, and for the self-relaxation from the protons near the spin probe to the bath (i.e. "lattice"), respectively. Enhancement vs. power (E(p)) curves acquired on cw ODNP instrumentation can quantify the cross-relaxivity (kσ) for concentrations as low as tens of micromolar. Furthermore, such data can include a correction for the microwave heating effects previously mentioned. Independent measurements can provide accurate values for the self-relaxivity (kρ) that are not affected by microwave heating, and which will have even further improved accuracy when obtained from samples of larger volume or higher concentration. The more accurate value for the coupling factor, ξ, that results from this new technique more reliably quantifies the local translational diffusivity, D local , near the spin probe and opens up the novel possibility of analyzing lower sample concentrations of ≤ 100 µM that are critical for biomolecular studies. To demonstrate these improvements and compare to recent results, we repeat careful measurements of the coupling factor (ξ) between a small nitroxide probe (4-hydroxy-TEMPO) and otherwise unperturbed bulk water, at both high and low spin probe concentrations. At high concentrations, we measure a significantly higher extrapolated enhancement, Emax, than was previously measured or predicted by solely cw ODNP-based work [6]. At all concentrations, for the first time, the data measured by the cw ODNP instrumentation shown here agrees with the coupling factor values of 0.36 [1], 0.33-0.35 [12], or 0.33 [10, 11] that others have reported based on ODNP measurements augmented by FCR experiments and pulsed ESR experiments, or the value of 0.30 predicted by molecular dynamics simulations [13]. On the one hand, this observation resolves the debate revolving around the absolute value of the coupling factor between water and freely dissolved spin probes, which is an important reference value for the study of hydration water in biological and other macromolecular systems. Our data conclusively supports a values of 0.33 [10, 11] rather than 0.22 [1, 6]. On the other hand, contrary to conclusions drawn in previous literature [11, 14], this data implies that solely cw ODNP methods can provide quantitative and accurate coupling factors, and thus derive accurate hydration dynamics information. This is fortuitous; FCR and pulsed ESR tools will continue to present powerful and complementary capabilities, while the implementation of quantitative ODNP measurements on widely available and easy to use cw ODNP instrumentation has distinctly practical benefits for the end user.

Over recent decades, motivated either by practicality or the need to tap into new types of measur... more Over recent decades, motivated either by practicality or the need to tap into new types of measurements, the science of Magnetic Resonance has expanded into more adverse conditions: deliberately chosen lower frequencies, inhomogeneous fields, and/or time-variable fields. In particular, Overhauser Dynamic Nuclear Polarization (ODNP) presents a case study that challenges previous expectations and offers an interesting test-bed for further developments. For example, an interest in the nanoscale heterogeneities of hydration dynamics demand increasingly sophisticated and automated measurements deploying ODNP on a modular, open source instrument operating at 15 MHz. ODNP demands the acquisition and automated processing of large quantities of one dimensional NMR spectra, which can present various problems: in particular, unambiguous identification of signal in newly configured instruments presents a practical challenge, while field drift tends to remain an issue even in fully configured in...