Deuterium spin relaxation of fractionally deuterated ribonuclease H using paired 475 and 950 MHz NMR spectrometers.

Deuterium (2H) spin relaxation of 13CH2D methyl groups has been widely applied to investigate picosecond-to-nanosecond conformational dynamics in proteins by solution-state NMR spectroscopy. The B0 dependence of the 2H spin relaxation rates is represented by a linear relationship between the spectral density function at three discrete frequencies J(0), J(ωD) and J(2ωD). In this study, the linear relation between 2H relaxation rates at B0 fields separated by a factor of two and the interpolation of rates at intermediate frequencies are combined for a more robust approach for spectral density mapping. The general usefulness of the approach is demonstrated on a fractionally deuterated (55%) and alternate 13C-12C labeled sample of E. coli RNase H. Deuterium relaxation rate constants (R1, R1ρ, RQ, RAP) were measured for 57 well-resolved 13CH2D moieties in RNase H at 1H frequencies of 475 MHz, 500 MHz, 900 MHz, and 950 MHz. The spectral density mapping of the 475/950 MHz data combination was performed independently and jointly to validate the expected relationship between data recorded at B0 fields separated by a factor of two. The final analysis was performed by jointly analyzing 475/950 MHz rates with 700 MHz rates interpolated from 500/900 MHz data to yield six J(ωD) values for each methyl peak. The J(ω) profile for each peak was fit to the original (τM, Sf2, τf) or extended model-free function (τM, Sf2, Ss2, τf, τs) to obtain optimized dynamic parameters.

Comparisons of Ribonuclease HI Homologs and Mutants Uncover a Multistate Model for Substrate Recognition.

Ribonuclease HI (RNHI) nonspecifically cleaves the RNA strand of RNA:DNA hybrid duplexes in a myriad of biological processes. Several RNHI homologs contain an extended domain, termed the handle region, which is critical to substrate binding. Nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have suggested a kinetic model in which the handle region can exist in open (substrate-binding competent) or closed (substrate-binding incompetent) states in homologs containing arginine or lysine at position 88 (using sequence numbering of E. coli RNHI), while the handle region populates states intermediate between the open and closed conformers in homologs with asparagine at residue 88 [Stafford, K. A., et al., PLoS Comput. Biol. 2013, 9, 1-10]. NMR parameters characterizing handle region dynamics are highly correlated with enzymatic activity for RNHI homologs with two-state (open/closed) handle regions [Martin, J. A., et al., Biochemistry 2020, 59, 3201-3205]. The work presented herein shows that homologs containing asparagine 88 display distinct structural features compared with their counterparts containing arginine or lysine 88. Comparisons of RNHI homologs and site-directed mutants with asparagine 88 support a kinetic model for handle region dynamics that includes 12 unique transitions between eight conformations. Overall, these findings present an example of the structure-function relationships of enzymes and spotlight the use of NMR spectroscopy and MD simulations in uncovering fine details of conformational preferences.

Quantifying the Relationship between Conformational Dynamics and Enzymatic Activity in Ribonuclease HI Homologues.

Ribonuclease HI (RNHI), a ubiquitous, non-sequence-specific endonuclease, cleaves the RNA strand in RNA/DNA hybrids. RNHI functions in replication and genome maintenance, and retroviral reverse transcriptases contain an essential ribonuclease H domain. Nuclear magnetic resonance (NMR) spectroscopy combined with molecular dynamics (MD) simulations suggests a model in which the extended handle region domain of Escherichia coli RNHI populates (substrate-binding-competent) "open" and (substrate-binding-incompetent) "closed" states, while the thermophilic Thermus thermophilus RNHI mainly populates the closed state at 300 K [Stafford, K. A., Robustelli, P., and Palmer, A. G., III (2013) PLoS Comput. Biol. 9, 1-10]. In addition, an in silico-designed mutant E. coli Val98Ala RNHI was predicted to populate primarily the closed state. The work presented here validates this model and confirms the predicted properties of the designed mutant. MD simulations suggest that the conformational preferences of the handle region correlate with the conformations of Trp85, Thr92, and Val101. NMR residual dipolar coupling constants, three-bond scalar coupling constants, and chemical shifts experimentally define the conformational states of these residues and hence of the handle domain. These NMR parameters correlate with the Michaelis constants for RNHI homologues, confirming the important role of the handle region in the modulation of substrate recognition and illustrating the power of NMR spectroscopy in dissecting the conformational preferences underlying enzyme function.

Detection of chemical exchange in methyl groups of macromolecules.

The zero- and double-quantum methyl TROSY Hahn-echo and the methyl 1H-1H dipole-dipole cross-correlation nuclear magnetic resonance experiments enable estimation of multiple quantum chemical exchange broadening in methyl groups in proteins. The two relaxation rate constants are established to be linearly dependent using molecular dynamics simulations and empirical analysis of experimental data. This relationship allows chemical exchange broadening to be recognized as an increase in the Hahn-echo relaxation rate constant. The approach is illustrated by analyzing relaxation data collected at three temperatures for E. coli ribonuclease HI and by analyzing relaxation data collected for different cofactor and substrate complexes of E. coli AlkB.

Enhanced spectral density mapping through combined multiple-field deuterium 13CH2D methyl spin relaxation NMR spectroscopy.

Quadrupolar relaxation of 2H (D) nuclear spins is a powerful probe of conformational dynamics in biological macromolecules. Deuterium relaxation rate constants are determined by the spectral density function for reorientation of the C-D bond vector at zero, single-quantum, and double-quantum 2H frequencies. In the present work, 2H relaxation rate constants were measured for an E. coli ribonuclease H [U-2H, 15N] ILV-[13CH2D] sample using 400, 500, 800, and 900 MHz NMR spectrometers and analyzed by three approaches to determine spectral density values. First, data recorded at each static magnetic field were analyzed independently. Second, data recorded at 400 and 800 MHz were analyzed jointly and data recorded at other fields were analyzed independently. Third, data recorded at 400 and 500 MHz were interpolated to 450 MHz, and the resulting two pairs of data, corresponding to 400 MHz/800 MHz and 450 MHz/900 MHz, were analyzed jointly. The second and third approaches rely on the identity between the double quantum frequency at the lower field and the single quantum frequency at the higher field. Spectral density values for 32 of the 48 resolvable ILV methyl resonances were fit by the Lipari-Szabo model-free formalism and used to validate the three methods. The three spectral density mapping methods performed equally well in cross validation with data recorded at 700 MHz. However, the third method yielded approximately 10-15% more precise estimates of model-free parameters and consequently provides a general strategy for analysis of 2H spin relaxation data in biological macromolecules.

Analysis of NMR Spin-Relaxation Data Using an Inverse Gaussian Distribution Function.

Spin relaxation in solution-state NMR spectroscopy is a powerful approach to explore the conformational dynamics of biological macromolecules. Probability distribution functions for overall or internal correlation times have been used previously to model spectral density functions central to spin-relaxation theory. Applications to biological macromolecules rely on transverse relaxation rate constants, and when studying nanosecond timescale motions, sampling at ultralow frequencies is often necessary. Consequently, appropriate distribution functions necessitate spectral density functions that are accurate and convergent as frequencies approach zero. In this work, the inverse Gaussian probability distribution function is derived from general properties of spectral density functions at low and high frequencies for macromolecules in solution, using the principle of maximal entropy. This normalized distribution function is first used to calculate the correlation function, followed by the spectral density function. The resulting model-free spectral density functions are finite at a frequency of zero and can be used to describe distributions of either overall or internal correlation times using the model-free ansatz. To validate the approach, 15N spin-relaxation data for the bZip transcription factor domain of the Saccharomyces cerevisiae protein GCN4, in the absence of cognate DNA, were analyzed using the inverse Gaussian probability distribution for intramolecular correlation times. The results extend previous models for the conformational dynamics of the intrinsically disordered, DNA-binding region of the bZip transcription factor domain.

General Expressions for Carr-Purcell-Meiboom-Gill Relaxation Dispersion for N-Site Chemical Exchange.

The Carr-Purcell-Meiboom-Gill (CPMG) nuclear magnetic resonance experiment is widely used to characterize chemical exchange phenomena in biological macromolecules. Theoretical expressions for the nuclear spin relaxation rate constant for two-site chemical exchange during CPMG pulse trains valid for all time scales are well-known as are descriptions of N-site exchange in the fast limit. We have obtained theoretical expressions for N-site exchange outside of the fast limit by using approximations to an average Liouvillian describing the decay of magnetization during a CPMG pulse train. We obtain general expressions for CPMG experiments for any N-site scheme and all experimentally accessible time scales. For sufficiently slow chemical exchange, we obtain closed-form expressions for the relaxation rate constant and a general characteristic polynomial for arbitrary kinetic schemes. Furthermore, we highlight features that qualitatively characterize CPMG curves obtained for various N-site kinetic topologies, quantitatively characterize CPMG curves obtained from systems in various N-site exchange situations, and test distinguishability of kinetic models.

TROSY pulse sequence for simultaneous measurement of the 15N R1 and {1H}-15N NOE in deuterated proteins.

A TROSY-based NMR experiment is described for simultaneous measurement of the 15N longitudinal relaxation rate constant R1 and the {1H}-15N nuclear Overhauser enhancement. The experiment is based on the observation that the TROSY mixing pulse sequence element symmetrically exchanges 1H and 15N magnetizations. The accuracy of the proposed technique is validated by comparison to independent measurements of both relaxation parameters for the protein ubiquitin. The simultaneous experiment is approximately 20-33% shorter than conventional sequential measurements.

Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective.

Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is up-regulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI's role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective lead optimized compound (LOC)14, that acts as a modulator of PDI. LOC14 was identified using a high-throughput screen of ∼10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a Kd of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease.

Enzyme dynamics from NMR spectroscopy.

CONSPECTUS: Biological activities of enzymes, including regulation or coordination of mechanistic stages preceding or following the chemical step, may depend upon kinetic or equilibrium changes in protein conformations. Exchange of more open or flexible conformational states with more closed or constrained states can influence inhibition, allosteric regulation, substrate recognition, formation of the Michaelis complex, side reactions, and product release. NMR spectroscopy has long been applied to the study of conformational dynamic processes in enzymes because these phenomena can be characterized over multiple time scales with atomic site resolution. Laboratory-frame spin-relaxation measurements, sensitive to reorientational motions on picosecond-nanosecond time scales, and rotating-frame relaxation-dispersion measurements, sensitive to chemical exchange processes on microsecond-millisecond time scales, provide information on both conformational distributions and kinetics. This Account reviews NMR spin relaxation studies of the enzymes ribonuclease HI from mesophilic (Escherichia coli) and thermophilic (Thermus thermophilus) bacteria, E. coli AlkB, and Saccharomyces cerevisiae triosephosphate isomerase to illustrate the contributions of conformational flexibility and dynamics to diverse steps in enzyme mechanism. Spin relaxation measurements and molecular dynamics (MD) simulations of the bacterial ribonuclease H enzymes show that the handle region, one of three loop regions that interact with substrates, interconverts between two conformations. Comparison of these conformations with the structure of the complex between Homo sapiens ribonuclease H and a DNA:RNA substrate suggests that the more closed state is inhibitory to binding. The large population of the closed conformation in T. thermophilus ribonuclease H contributes to the increased Michaelis constant compared with the E. coli enzyme. NMR spin relaxation and fluorescence spectroscopy have characterized a conformational transition in AlkB between an open state, in which the side chains of methionine residues in the active site are disordered, and a closed state, in which these residues are ordered. The open state is highly populated in the AlkB/Zn(II) complex, and the closed state is highly populated in the AlkB/Zn(II)/2OG/substrate complex, in which 2OG is the 2-oxoglutarate cosubstrate and the substrate is a methylated DNA oligonucleotide. The equilibrium is shifted to approximately equal populations of the two conformations in the AlkB/Zn(II)/2OG complex. The conformational shift induced by 2OG ensures that 2OG binds to AlkB/Zn(II) prior to the substrate. In addition, the opening rate of the closed conformation limits premature release of substrate, preventing generation of toxic side products by reaction with water. Closure of active site loop 6 in triosephosphate isomerase is critical for forming the Michaelis complex, but reopening of the loop after the reaction is (partially) rate limiting. NMR spin relaxation and MD simulations of triosephosphate isomerase in complex with glycerol 3-phosphate demonstrate that closure of loop 6 is a highly correlated rigid-body motion. The MD simulations also indicate that motions of Gly173 in the most flexible region of loop 6 contribute to opening of the active site loop for product release. Considered together, these three enzyme systems illustrate the power of NMR spin relaxation investigations in providing global insights into the role of conformational dynamic processes in the mechanisms of enzymes from initial activation to final product release.

Dynamics of GCN4 facilitate DNA interaction: a model-free analysis of an intrinsically disordered region.

Intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) are known to play important roles in regulatory and signaling pathways. A critical aspect of these functions is the ability of IDP/IDRs to form highly specific complexes with target molecules. However, elucidation of the contributions of conformational dynamics to function has been limited by challenges associated with structural heterogeneity of IDP/IDRs. Using NMR spin relaxation parameters ((15)N R1, (15)N R2, and {(1)H}-(15)N heteronuclear NOE) collected at four static magnetic fields ranging from 14.1 to 21.1 T, we have analyzed the backbone dynamics of the basic leucine-zipper (bZip) domain of the Saccharomyces cerevisiae transcription factor GCN4, whose DNA binding domain is intrinsically disordered in the absence of DNA substrate. We demonstrate that the extended model-free analysis can be applied to proteins with IDRs such as apo GCN4 and that these results significantly extend previous NMR studies of GCN4 dynamics performed using a single static magnetic field of 11.74 T [Bracken, et al., J. Mol. Biol., 1999, 285, 2133-2146] and correlate well with molecular dynamics simulations [Robustelli, et al., J. Chem. Theory Comput., 2013, 9, 5190-5200]. In contrast to the earlier work, data at multiple static fields allows the time scales of internal dynamics of GCN4 to be reliably quantified. Large amplitude dynamic fluctuations in the DNA-binding region have correlation times (τs ≈ 1.4-2.5 ns) consistent with a two-step mechanism in which partially ordered bZip conformations of GCN4 form initial encounter complexes with DNA and then rapidly rearrange to the high affinity state with fully formed basic region recognition helices.

Mechanism of E-cadherin dimerization probed by NMR relaxation dispersion.

Epithelial cadherin (E-cadherin), a member of the classical cadherin family, mediates calcium-dependent homophilic cell-cell adhesion. Crystal structures of classical cadherins reveal an adhesive dimer interface featuring reciprocal exchange of N-terminal ß-strands between two protomers. Previous work has identified a putative intermediate (called the "X-dimer") in the dimerization pathway of wild-type E-cadherin EC1-EC2 domains, based on crystal structures of mutants not capable of strand swapping and on deceleration of binding kinetics by mutations at the X-dimer interface. In the present work, NMR relaxation dispersion spectroscopy is used to directly observe and characterize intermediate states without the need to disrupt the strand-swapped binding interface by mutagenesis. The results indicate that E-cadherin forms strand-swapped dimers predominantly by a mechanism in which formation of a weak and short-lived X-dimer-like state precedes the conformational changes required for formation of the mature strand-swapped dimeric structure. Disruption of this intermediate state through mutation reduces both association and dissociation rates by factors of ~10(4), while minimally perturbing affinity. The X-dimer interface lowers the energy barrier associated with strand swapping and enables E-cadherins to form strand-swapped dimers at a rate consistent with residence times in adherens junctions.

Protein dynamics control the progression and efficiency of the catalytic reaction cycle of the Escherichia coli DNA-repair enzyme AlkB.

A central goal of enzymology is to understand the physicochemical mechanisms that enable proteins to catalyze complex chemical reactions with high efficiency. Recent methodological advances enable the contribution of protein dynamics to enzyme efficiency to be explored more deeply. Here, we utilize enzymological and biophysical studies, including NMR measurements of conformational dynamics, to develop a quantitative mechanistic scheme for the DNA repair enzyme AlkB. Like other iron/2-oxoglutarate-dependent dioxygenases, AlkB employs a two-step mechanism in which oxidation of 2-oxoglutarate generates a highly reactive enzyme-bound oxyferryl intermediate that, in the case of AlkB, slowly hydroxylates an alkylated nucleobase. Our results demonstrate that a microsecond-to-millisecond time scale conformational transition facilitates the proper sequential order of substrate binding to AlkB. Mutations altering the dynamics of this transition allow generation of the oxyferryl intermediate but promote its premature quenching by solvent, which uncouples 2-oxoglutarate turnover from nucleobase oxidation. Therefore, efficient catalysis by AlkB depends upon the dynamics of a specific conformational transition, establishing another paradigm for the control of enzyme function by protein dynamics.

Conformational flexibility in the binding surface of the potassium channel blocker ShK.

ShK is a 35-residue peptide that binds with high affinity to human voltage-gated potassium channels through a conserved K-Y dyad. Here we have employed NMR measurements of backbone-amide (15)N spin-relaxation rates to investigate motions of the ShK backbone. Although ShK is rigid on the ps to ns timescale, increased linewidths observed for 11 backbone-amide (15)N resonances identify chemical or conformational exchange contributions to the spin relaxation. Relaxation dispersion profiles indicate that exchange between major and minor conformers occurs on the sub-millisecond timescale. Affected residues are mostly clustered around the central helix-kink-helix structure and the critical K22-Y23 motif. We suggest that the less structured minor conformer increases the exposure of Y23, known to contribute to binding affinity and selectivity, thereby facilitating its interaction with potassium channels. These findings have potential implications for the design of new channel blockers based on ShK.

Thermal adaptation of conformational dynamics in ribonuclease H.

The relationship between inherent internal conformational processes and enzymatic activity or thermodynamic stability of proteins has proven difficult to characterize. The study of homologous proteins with differing thermostabilities offers an especially useful approach for understanding the functional aspects of conformational dynamics. In particular, ribonuclease HI (RNase H), an 18 kD globular protein that hydrolyzes the RNA strand of RNA:DNA hybrid substrates, has been extensively studied by NMR spectroscopy to characterize the differences in dynamics between homologs from the mesophilic organism E. coli and the thermophilic organism T. thermophilus. Herein, molecular dynamics simulations are reported for five homologous RNase H proteins of varying thermostabilities and enzymatic activities from organisms of markedly different preferred growth temperatures. For the E. coli and T. thermophilus proteins, strong agreement is obtained between simulated and experimental values for NMR order parameters and for dynamically averaged chemical shifts, suggesting that these simulations can be a productive platform for predicting the effects of individual amino acid residues on dynamic behavior. Analyses of the simulations reveal that a single residue differentiates between two different and otherwise conserved dynamic processes in a region of the protein known to form part of the substrate-binding interface. Additional key residues within these two categories are identified through the temperature-dependence of these conformational processes.

Breaking down walls: Continuous potential models for internal motions in NMR spin relaxation.

Simple physical models for restricted diffusion in a potential, which provide important insights for NMR spin relaxation, usually are based on free diffusion within rigid boundaries or diffusion in relatively simple continuous potential energy surfaces. The diffusion-in-a-cone model is an example of the former and diffusion in an N-fold cosine potential is an example of the latter. The present work models restricted diffusion for arbitrary potential energy functions on the surface of a cone or a sphere, by expanding the potentials in Fourier or spherical harmonic series, respectively. The results exhibit simple relationships between generalized order parameters and effective correlation times, critical for analysis of experimental spin relaxation data, and illustrate the transition from diffusive-like to jump-like behavior in multi-well potentials.

Parsing Dynamics of Protein Backbone NH and Side-Chain Methyl Groups using Molecular Dynamics Simulations.

Experimental NMR spectroscopy and theoretical molecular dynamics (MD) simulations provide complementary insights into protein conformational dynamics and hence into biological function. The present work describes an extensive set of backbone NH and side-chain methyl group generalized order parameters for the Escherichia coli ribonuclease HI (RNH) enzyme derived from 2-µs microsecond MD simulations using the OPLS4 and AMBER-FF19SB force fields. The simulated generalized order parameters are compared with values derived from NMR 15N and 13CH2D spin relaxation measurements. The squares of the generalized order parameters, S2 for the N-H bond vector and Saxis2 for the methyl group symmetry axis, characterize the equilibrium distribution of vector orientations in a molecular frame of reference. Optimal agreement between simulated and experimental results was obtained by averaging S2 or Saxis2 calculated by dividing the simulated trajectories into 50 ns blocks (∼five times the rotational diffusion correlation time for RNH). With this procedure, the median absolute deviations (MAD) between experimental and simulated values of S2 and Saxis2 are 0.030 (NH) and 0.061 (CH3) for OPLS4 and 0.041 (NH) and 0.078 (CH3) for AMBER-FF19SB. The MAD between OPLS4 and AMBER-FF19SB are 0.021 (NH) and 0.072 (CH3). The generalized order parameters for the methyl group symmetry axis can be decomposed into contributions from backbone fluctuations, between-rotamer dihedral angle transitions, and within-rotamer dihedral angle fluctuations. Analysis of the simulation trajectories shows that (i) backbone and side chain conformational fluctuations exhibit little correlation and that (ii) fluctuations within rotamers are limited and highly uniform with values that depend on the number of dihedral angles considered. Low values of Saxis2, indicative of enhanced side-chain flexibility, result from between-rotamer transitions that can be enhanced by increased local backbone flexibility.

LC-Photo-CIDNP hyperpolarization of biomolecules bearing a quasi-isolated spin pair: Magnetic-Field dependence via a rapid-shuttling device.

Liquid-state low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) is an emerging technology tailored to enhance the sensitivity of NMR spectroscopy via LED- or laser-mediated optical irradiation. LC-photo-CIDNP is particularly useful to detect solvent-exposed aromatic residues (Trp, Tyr), either in isolation or within polypeptides and proteins. This study investigates the magnetic-field dependence of the LC-photo-CIDNP of Trp-α-13C-ß,ß,2,4,5,6,7-d7, a Trp isotopolog bearing a quasi-isolated 1Hα-13Cαspin pair (QISP). We employed a new rapid-shuttling side-illumination field-cycling device that enables ultra-fast (90-120 ms) vertical movements of NMR samples within the bore of a superconducting magnet. Thus, LC-photo-CIDNP hyperpolarization occurs at low field, while hyperpolarized signals are detected at high field (700 MHz). Resonance lineshapes were excellent, and the effect of several fields (1.18-7.08 T range) on hyperpolarization efficiency could be readily explored. Remarkably, unprecedented LC-photo-CIDNP enhancements ε ≅ 1,200 were obtained at 50 MHz (1.18 T), suggesting exciting avenues to hypersensitive LED-enhanced NMR in liquids at low field.