Nathaniel V. Nucci

Site-resolved measurement of water-protein interactions by solution NMR

The interactions of biological macromolecules with water are fundamental to their structure, dynamics and function. Historically, characterization of the location and residence times of hydration waters of proteins in solution has been quite difficult. Confining proteins within the nanoscale interior of a reverse micelle slows water dynamics, allowing global protein-water interactions to be detected using nuclear magnetic resonance techniques. Complications that normally arise from hydrogen exchange and long-range dipolar coupling are overcome by the nature of the reverse micelle medium. Characterization of the hydration of ubiquitin demonstrates that encapsulation within a reverse micelle allows detection of dozens of hydration waters. Comparison of nuclear Overhauser effects obtained in the laboratory and rotating frames indicate a considerable range of hydration water dynamics is present on the protein surface. In addition, an unprecedented clustering of different hydration-dynamics classes of sites is evident.

Mapping the hydration dynamics of ubiquitin.

The nature of waters interaction with biomolecules such as proteins has been difficult to examine in detail at atomic resolution. Solution NMR spectroscopy is potentially a powerful method for characterizing both the structural and temporal aspects of protein hydration but has been plagued by artifacts. Encapsulation of the protein of interest within the aqueous core of a reverse micelle particle results in a general slowing of water dynamics, significant reduction in hydrogen exchange chemistry and elimination of contributions from bulk water thereby enabling the use of nuclear Overhauser effects to quantify interactions between the protein surface and hydration water. Here we extend this approach to allow use of dipolar interactions between hydration water and hydrogens bonded to protein carbon atoms. By manipulating the molecular reorientation time of the reverse micelle particle through use of low viscosity liquid propane, the T(1ρ) relaxation time constants of (1)H bonded to (13)C were sufficiently lengthened to allow high quality rotating frame nuclear Overhauser effects to be obtained. These data supplement previous results obtained from dipolar interactions between the protein and hydrogens bonded to nitrogen and in aggregate cover the majority of the molecular surface of the protein. A wide range of hydration dynamics is observed. Clustering of hydration dynamics on the molecular surface is also seen. Regions of long-lived hydration water correspond with regions of the protein that participate in molecular recognition of binding partners suggesting that the contribution of the solvent entropy to the entropy of binding has been maximized through evolution.

Changes in water structure induced by the guanidinium cation and implications for protein denaturation.

The effect of the guanidinium cation on the hydrogen bonding strength of water was analyzed using temperature-excursion Fourier transform infrared spectra of the OH stretching vibration in 5% H 2O/95% D 2O solutions containing a range of different guanidine-HCl and guanidine-HBr concentrations. Our findings indicate that the guanidinium cation causes the water H-bonds in solution to become more linear than those found in bulk water, and that it also inhibits the response of the H-bond network to increased temperature. Quantum chemical calculations also reveal that guanidinium affects both the charge distribution on water molecules directly H-bonded to it as well as the OH stretch frequency of H-bonds in which that water molecule is the donor. The implications of our findings to hydrophobic solvation and protein denaturation are discussed.

Role of cavities and hydration in the pressure unfolding of T4 lysozyme.

Significance Pressure unfolding of proteins is a fundamental aspect of their thermodynamic response, the origins of which remain controversial. Here, we use high-pressure solution NMR to investigate the pressure response of a model protein, T4 lysozyme, under various conditions. Our data resolve longstanding controversies regarding the pressure response of this protein and the hydration of the internal hydrophobic cavity. It is shown that local packing (cavities) and the availability of conformational space have important and nonlocal impacts on the protein pressure response. Overall, the findings presented here reveal a previously unappreciated complexity in the pressure response of protein structure. It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A–benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.

Optimized reverse micelle surfactant system for high-resolution NMR spectroscopy of encapsulated proteins and nucleic acids dissolved in low viscosity fluids.

An optimized reverse micelle surfactant system has been developed for solution nuclear magnetic resonance studies of encapsulated proteins and nucleic acids dissolved in low viscosity fluids. Comprising the nonionic 1-decanoyl-rac-glycerol and the zwitterionic lauryldimethylamine-N-oxide (10MAG/LDAO), this mixture is shown to efficiently encapsulate a diverse set of proteins and nucleic acids. Chemical shift analyses of these systems show that high structural fidelity is achieved upon encapsulation. The 10MAG/LDAO surfactant system reduces the molecular reorientation time for encapsulated macromolecules larger than ∼20 kDa leading to improved overall NMR performance. The 10MAG/LDAO system can also be used for solution NMR studies of lipid-modified proteins. New and efficient strategies for optimization of encapsulation conditions are described. 10MAG/LDAO performs well in both the low viscosity pentane and ultralow viscosity liquid ethane and therefore will serve as a general surfactant system for initiating solution NMR studies of proteins and nucleic acids.

Defining the Apoptotic Trigger: THE INTERACTION OF CYTOCHROME c AND CARDIOLIPIN.

Background: Binding of mitochondrial cardiolipin to cytochrome c is thought to trigger apoptosis. Results: Reverse micelle encapsulation mimics the confinement of the inner mitochrondrial membrane allowing binding of cardiolipin to be followed by NMR at atomic resolution. Conclusion: Cardiolipin interacts with cytochrome c without causing protein unfolding. Significance: A new model for the role of cytochrome c in apoptosis is required. The interaction between cytochrome c and the anionic lipid cardiolipin has been proposed as a primary event in the apoptotic signaling cascade. Numerous studies that have examined the interaction of cytochrome c with cardiolipin embedded in a variety of model phospholipid membranes have suggested that partial unfolding of the protein is a precursor to the apoptotic response. However, these studies lacked site resolution and used model systems with negligible or a positive membrane curvature, which is distinct from the large negative curvature of the invaginations of the inner mitochondrial membrane where cytochrome c resides. We have used reverse micelle encapsulation to mimic the potential effects of confinement on the interaction of cytochrome c with cardiolipin. Encapsulation of oxidized horse cytochrome c in 1-decanoyl-rac-glycerol/lauryldimethylamine-N-oxide/hexanol reverse micelles prepared in pentane yields NMR spectra essentially identical to the protein in free aqueous solution. The structure of encapsulated ferricytochrome c was determined to high precision (bb ∼ 0.23 Å) using NMR-based methods and is closely similar to the cryogenic crystal structure (bb ∼ 1.2 Å). Incorporation of cardiolipin into the reverse micelle surfactant shell causes localized chemical shift perturbations of the encapsulated protein, providing the first view of the cardiolipin/cytochrome c interaction interface at atomic resolution. Three distinct sites of interaction are detected: the so-called A- and L-sites, plus a previously undocumented interaction centered on residues Phe-36, Gly-37, Thr-58, Trp-59, and Lys-60. Importantly, in distinct contrast to earlier studies of this interaction, the protein is not significantly disturbed by the binding of cardiolipin in the context of the reverse micelle.

Measurement and Control of pH in the Aqueous Interior of Reverse Micelles

The encapsulation of proteins and nucleic acids within the nanoscale water core of reverse micelles has been used for over 3 decades as a vehicle for a wide range of investigations including enzymology, the physical chemistry of confined spaces, protein and nucleic acid structural biology, and drug development and delivery. Unfortunately, the static and dynamical aspects of the distribution of water in solutions of reverse micelles complicate the measurement and interpretation of fundamental parameters such as pH. This is a severe disadvantage in the context of (bio)chemical reactions and protein structure and function, which are generally highly sensitive to pH. There is a need to more fully characterize and control the effective pH of the reverse micelle water core. The buffering effect of titratable head groups of the reverse micelle surfactants is found to often be the dominant variable defining the pH of the water core. Methods for measuring the pH of the reverse micelle aqueous interior using one-dimensional 1H and two-dimensional heteronuclear NMR spectroscopy are described. Strategies for setting the effective pH of the reverse micelle water core are demonstrated. The exquisite sensitivity of encapsulated proteins to the surfactant, water content, and pH of the reverse micelle is also addressed. These results highlight the importance of assessing the structural fidelity of the encapsulated protein using multidimensional NMR before embarking upon a detailed structural and biophysical characterization.

Pyrene-1-Carboxylate in Water and Glycerol Solutions: Origin of the Change of pK Upon Excitation

Pyrene-1-carboxy acid has a pK of 4 in the ground state, and a pK of 8 in the excited state. Fluorescence spectra of the acid and base forms are presented as a function of solvent and temperature. Ab initio quantum calculations indicate that the bond between the ring system and the carboxyl group has aromatic character that becomes stronger upon excitation. This stabilization helps to account for the increase in pK upon excitation.

Reverse micelles as a platform for dynamic nuclear polarization in solution NMR of proteins.

Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to ∼−93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other biomolecules.

Modification of encapsulation pressure of reverse micelles in liquid ethane

Encapsulation within reverse micelles dissolved in low viscosity fluids offers a potential solution to the slow tumbling problem presented by large soluble macromolecules to solution NMR spectroscopy. The reduction in effective macromolecular tumbling is directly dependent upon the viscosity of the solvent. Liquid ethane is of sufficiently low viscosity at pressures below 5000 psi to offer a significant advantage. Unfortunately, the viscosity of liquid ethane shows appreciable pressure dependence. Reverse micelle encapsulation in liquid ethane often requires significantly higher pressures, which obviates the potential advantages offered by liquid ethane over liquid propane. Addition of co-surfactants or co-solvents can be used to manipulate the minimum pressure required to obtain stable, well-behaved solutions of reverse micelles prepared in liquid ethane. A library of potential additives is examined and several candidates suitable for use with encapsulated proteins are described.