Unraveling motion in proteins by combining NMR relaxometry and molecular dynamics simulations: A case study on ubiquitin.

Nuclear magnetic resonance (NMR) relaxation experiments shine light onto the dynamics of molecular systems in the picosecond to millisecond timescales. As these methods cannot provide an atomically resolved view of the motion of atoms, functional groups, or domains giving rise to such signals, relaxation techniques have been combined with molecular dynamics (MD) simulations to obtain mechanistic descriptions and gain insights into the functional role of side chain or domain motion. In this work, we present a comparison of five computational methods that permit the joint analysis of MD simulations and NMR relaxation experiments. We discuss their relative strengths and areas of applicability and demonstrate how they may be utilized to interpret the dynamics in MD simulations with the small protein ubiquitin as a test system. We focus on the aliphatic side chains given the rigidity of the backbone of this protein. We find encouraging agreement between experiment, Markov state models built in the χ1/χ2 rotamer space of isoleucine residues, explicit rotamer jump models, and a decomposition of the motion using ROMANCE. These methods allow us to ascribe the dynamics to specific rotamer jumps. Simulations with eight different combinations of force field and water model highlight how the different metrics may be employed to pinpoint force field deficiencies. Furthermore, the presented comparison offers a perspective on the utility of NMR relaxation to serve as validation data for the prediction of kinetics by state-of-the-art biomolecular force fields.

Efficient 18.8 T MAS-DNP NMR reveals hidden side chains in amyloid fibrils.

Amyloid fibrils are large and insoluble protein assemblies composed of a rigid core associated with a cross-ß arrangement rich in ß-sheet structural elements. It has been widely observed in solid-state NMR experiments that semi-rigid protein segments or side chains do not yield easily observable NMR signals at room temperature. The reasons for the missing peaks may be due to the presence of unfavorable dynamics that interfere with NMR experiments, which result in very weak or unobservable NMR signals. Therefore, for amyloid fibrils, semi-rigid and dynamically disordered segments flanking the amyloid core are very challenging to study. Here, we show that high-field dynamic nuclear polarization (DNP), an NMR hyperpolarization technique typically performed at low temperatures, can circumvent this issue because (i) the low-temperature environment (~ 100 K) slows down the protein dynamics to escape unfavorable detection regime, (ii) DNP improves the overall NMR sensitivity including those of flexible side chains, and (iii) efficient cross-effect DNP biradicals (SNAPol-1) optimized for high-field DNP (≥ 18.8 T) are employed to offer high sensitivity and resolution suitable for biomolecular NMR applications. By combining these factors, we have successfully established an impressive enhancement factor of ε ~ 50 on amyloid fibrils using an 18.8 T/ 800 MHz magnet. We have compared the DNP efficiencies of M-TinyPol, NATriPol-3, and SNAPol-1 biradicals on amyloid fibrils. We found that SNAPol-1 (with ε ~ 50) outperformed the other two radicals. The MAS DNP experiments revealed signals of flexible side chains previously inaccessible at conventional room-temperature experiments. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains and dynamically disordered segments otherwise hidden at room temperature.

Convergent views on disordered protein dynamics from NMR and computational approaches.

Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) is a class of biologically important proteins exhibiting specific biophysical characteristics. They lack a hydrophobic core, and their conformational behavior is strongly influenced by electrostatic interactions. IDPs and IDRs are highly dynamic, and a characterization of the motions of IDPs and IDRs is essential for their physically correct description. NMR together with molecular dynamics simulations are the methods best suited to such a task because they provide information about dynamics of proteins with atomistic resolution. Here, we present a study of motions of a disordered C-terminal domain of the delta subunit of RNA polymerase from Bacillus subtilis. Positively and negatively charged residues in the studied domain form transient electrostatic contacts critical for the biological function. Our study is focused on investigation of ps-ns dynamics of backbone of the delta subunit based on analysis of amide 15N NMR relaxation data and molecular dynamics simulations. In order to extend an informational content of NMR data to lower frequencies, which are more sensitive to slower motions, we combined standard (high-field) NMR relaxation experiments with high-resolution relaxometry. Altogether, we collected data reporting the relaxation at 12 different magnetic fields, resulting in an unprecedented data set. Our results document that the analysis of such data provides a consistent description of dynamics and confirms the validity of so far used protocols of the analysis of dynamics of IDPs also for a partially folded protein. In addition, the potential to access detailed description of motions at the timescale of tens of ns with the help of relaxometry data is discussed. Interestingly, in our case, it appears to be mostly relevant for a region involved in the formation of temporary contacts within the disordered region, which was previously proven to be biologically important.

Explicit Models of Motion to Understand Protein Side-Chain Dynamics.

Nuclear magnetic relaxation is widely used to probe protein dynamics. For decades, most analyses of relaxation in proteins have relied successfully on the model-free approach, forgoing mechanistic descriptions of motion. Model-free types of correlation functions cannot describe a large carbon-13 relaxation dataset in protein side chains. Here, we use molecular dynamics simulations to design explicit models of motion and solve Fokker-Planck diffusion equations. These models of motion provide better agreement with relaxation data, mechanistic insight, and a direct link to configuration entropy.

Explicit models of motions to analyze NMR relaxation data in proteins.

Nuclear Magnetic Resonance (NMR) is a tool of choice to characterize molecular motions. In biological macromolecules, pico- to nanosecond motions, in particular, can be probed by nuclear spin relaxation rates, which depend on the time fluctuations of the orientations of spin interaction frames. For the past 40 years, relaxation rates have been successfully analyzed using the Model-Free (MF) approach, which makes no assumption on the nature of motions and reports on the effective amplitude and timescale of the motions. However, obtaining a mechanistic picture of motions from this type of analysis is difficult at best, unless complemented with molecular dynamics (MD) simulations. In spite of their limited accuracy, such simulations can be used to obtain the information necessary to build explicit models of motions designed to analyze NMR relaxation data. Here, we present how to build such models, suited in particular to describe motions of methyl-bearing protein side chains and compare them with the MF approach. We show on synthetic data that explicit models of motions are more robust in the presence of rotamer jumps which dominate the relaxation in methyl groups of protein side chains. We expect this work to motivate the use of explicit models of motion to analyze MD and NMR data.

Detection of Metabolite-Protein Interactions in Complex Biological Samples by High-Resolution Relaxometry: Toward Interactomics by NMR.

Metabolomics, the systematic investigation of metabolites in biological fluids, cells, or tissues, reveals essential information about metabolism and diseases. Metabolites have functional roles in a myriad of biological processes, as substrates and products of enzymatic reactions but also as cofactors and regulators of large numbers of biochemical mechanisms. These functions involve interactions of metabolites with macromolecules. Yet, methods to systematically investigate these interactions are still scarce to date. In particular, there is a need for techniques suited to identify and characterize weak metabolite-macromolecule interactions directly in complex media such as biological fluids. Here, we introduce a method to investigate weak interactions between metabolites and macromolecules in biological fluids. Our approach is based on high-resolution NMR relaxometry and does not require any invasive procedure or separation step. We show that we can detect interactions between small and large molecules in human blood serum and quantify the size of the complex. Our work opens the way for investigations of metabolite (or other small molecules)-protein interactions in biological fluids for interactomics or pharmaceutical applications.

How wide is the window opened by high-resolution relaxometry on the internal dynamics of proteins in solution?

The dynamics of molecules in solution is usually quantified by the determination of timescale-specific amplitudes of motions. High-resolution nuclear magnetic resonance (NMR) relaxometry experiments-where the sample is transferred to low fields for longitudinal (T1) relaxation, and back to high field for detection with residue-specific resolution-seeks to increase the ability to distinguish the contributions from motion on timescales slower than a few nanoseconds. However, tumbling of a molecule in solution masks some of these motions. Therefore, we investigate to what extent relaxometry improves timescale resolution, using the "detector" analysis of dynamics. Here, we demonstrate improvements in the characterization of internal dynamics of methyl-bearing side chains by carbon-13 relaxometry in the small protein ubiquitin. We show that relaxometry data leads to better information about nanosecond motions as compared to high-field relaxation data only. Our calculations show that gains from relaxometry are greater with increasing correlation time of rotational diffusion.

Sequential assignment of NMR spectra of peptides at natural isotopic abundance with zero- and ultra-low-field total correlation spectroscopy (ZULF-TOCSY).

A novel method dubbed ZULF-TOCSY results from the combination of Zero and Ultra-Low Field (ZULF) with high-field, high-resolution NMR, leading to a generalization of the concept of total correlation spectroscopy (TOCSY). ZULF-TOCSY is a new building block for NMR methods, which has the unique property that the polarization is evenly distributed among all NMR-active nuclei such as 1H, 13C, 15N, 31P, etc., provided that they belong to the same coupling network, and provided that their relaxation is not too fast at low fields, as may occur in macromolecules. Here, we show that ZULF-TOCSY correlations can be observed for peptides at natural isotopic abundance, such as the protected hexapeptide Boc-Met-enkephalin. The analysis of ZULF-TOCSY spectra readily allows one to make sequential assignments, thus offering an alternative to established heteronuclear 2D experiments like HMBC. For Boc-Met-enkephalin, we show that ZULF-TOCSY allows one to observe all expected cross-peaks between carbonyl carbons and α-CH protons, while the popular HMBC method provides insufficient information.

Boosting the resolution of low-field [Formula: see text] relaxation experiments on intrinsically disordered proteins with triple-resonance NMR.

Improving our understanding of nanosecond motions in disordered proteins requires the enhanced sampling of the spectral density function obtained from relaxation at low magnetic fields. High-resolution relaxometry and two-field NMR measurements of relaxation have, so far, only been based on the recording of one- or two-dimensional spectra, which provide insufficient resolution for challenging disordered proteins. Here, we introduce a 3D-HNCO-based two-field NMR experiment for measurements of protein backbone [Formula: see text] amide longitudinal relaxation rates. The experiment provides accurate longitudinal relaxation rates at low field (0.33 T in our case) preserving the resolution and sensitivity typical for high-field NMR spectroscopy. Radiofrequency pulses applied on six different radiofrequency channels are used to manipulate the spin system at both fields. The experiment was demonstrated on the C-terminal domain of [Formula: see text] subunit of RNA polymerase from Bacillus subtilis, a protein with highly repetitive amino-acid sequence and very low dispersion of backbone chemical shifts.

Sensitivity-enhanced three-dimensional and carbon-detected two-dimensional NMR of proteins using hyperpolarized water.

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

Reducing bias in the analysis of solution-state NMR data with dynamics detectors.

Nuclear magnetic resonance (NMR) is sensitive to dynamics on a wide range of correlation times. Recently, we have shown that analysis of relaxation rates via fitting to a correlation function with a small number of exponential terms could yield a biased characterization of molecular motion in solid-state NMR due to limited sensitivity of experimental data to certain ranges of correlation times. We introduced an alternative approach based on "detectors" in solid-state NMR, for which detector responses characterize motion for a range of correlation times and reduce potential bias resulting from the use of simple models for the motional correlation functions. Here, we show that similar bias can occur in the analysis of solution-state NMR relaxation data. We have thus adapted the detector approach to solution-state NMR, specifically separating overall tumbling motion from internal motions and accounting for contributions of chemical exchange to transverse relaxation. We demonstrate that internal protein motions can be described with detectors when the overall motion and the internal motions are statistically independent. We illustrate the detector analysis on ubiquitin with typical relaxation data sets recorded at a single high magnetic field or at multiple high magnetic fields and compare with results of model-free analysis. We also compare our methodology to LeMaster's method of dynamics analysis.

Understanding the methyl-TROSY effect over a wide range of magnetic fields.

The use of relaxation interference in the methyl Transverse Relaxation-Optimized SpectroscopY (TROSY) experiment has opened new avenues for the study of large proteins and protein assemblies in nuclear magnetic resonance. So far, the theoretical description of the methyl-TROSY experiment has been limited to the slow-tumbling approximation, which is correct for large proteins on high-field spectrometers. In a recent paper, favorable relaxation interference was observed in the methyl groups of a small protein at a magnetic field as low as 0.33 T, well outside the slow-tumbling regime. Here, we present a model to describe relaxation interference in methyl groups over a broad range of magnetic fields, not limited to the slow-tumbling regime. We predict that the type of multiple-quantum transition that shows favorable relaxation properties change with the magnetic field. Under the condition of fast methyl-group rotation, methyl-TROSY experiments can be recorded over the entire range of magnetic fields from a fraction of 1 T up to 100 T.

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.

Time-Resolved Protein Side-Chain Motions Unraveled by High-Resolution Relaxometry and Molecular Dynamics Simulations.

Motions of proteins are essential for the performance of their functions. Aliphatic protein side chains and their motions play critical roles in protein interactions: for recognition and binding of partner molecules at the surface or serving as an entropy reservoir within the hydrophobic core. Here, we present a new NMR method based on high-resolution relaxometry and high-field relaxation to determine quantitatively both motional amplitudes and time scales of methyl-bearing side chains in the picosecond-to-nanosecond range. We detect a wide variety of motions in isoleucine side chains in the protein ubiquitin. We unambiguously identify slow motions in the low nanosecond range, which, in conjunction with molecular dynamics computer simulations, could be assigned to transitions between rotamers. Our approach provides unmatched detailed insight into the motions of aliphatic side chains in proteins and provides a better understanding of the nature and functional role of protein side-chain motions.

High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents.

Hyperpolarized 2D exchange spectroscopy (HYPEX) to obtain high-resolution nuclear magnetic resonance (NMR) spectra of folded proteins under near-physiological conditions is reported. The technique is based on hyperpolarized water, which is prepared by dissolution dynamic nuclear polarization and mixed in situ in an NMR spectrometer with a protein in a physiological saline buffer at body temperature. Rapid exchange of labile protons with the hyperpolarized solvent, combined with cross-relaxation effects (NOEs), leads to boosted signal intensities for many amide 1 H-15 N correlations in the protein ubiquitin. As the introduction of hyperpolarization to the target protein is mediated via the solvent, the method is applicable to a broad spectrum of target molecules.

Structure and Dynamics of an Intrinsically Disordered Protein Region That Partially Folds upon Binding by Chemical-Exchange NMR.

Many intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-state NMR, signals of the IDR in the interacting region become broad, weak, and often invisible, while X-ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical exchange by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts, and site-specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins.

Full Correlations across Broad NMR Spectra by Two-Field Total Correlation Spectroscopy.

Total correlation spectroscopy (TOCSY) is a key experiment to assign nuclear magnetic resonance (NMR) spectra of complex molecules. Carbon-13 TOCSY experiments are essential to assign signals of protein side chains. However, the performance of carbon-13 TOCSY deteriorates at high magnetic fields since the necessarily limited radiofrequency irradiation fails to cover the broad range of carbon-13 frequencies. Here, we introduce a new concept to overcome the limitations of TOCSY by using two-field NMR spectroscopy. In two-field TOCSY experiments, chemical shifts are labelled at high field but isotropic mixing is performed at a much lower magnetic field, where the frequency range of the spectrum is drastically reduced. We obtain complete correlations between all carbon-13 nuclei belonging to amino acids across the entire spectrum: aromatic, aliphatic and carboxylic. Two-field TOCSY should be a robust and general approach for the assignment of uniformly carbon-13 labelled molecules in high-field and ultra-high field NMR spectrometers beyond 1000 MHz.

Protein dynamics from nuclear magnetic relaxation.

Nuclear magnetic resonance is a ubiquitous spectroscopic tool to explore molecules with atomic resolution. Nuclear magnetic relaxation is intimately connected to molecular motions. Many methods and models have been developed to measure and interpret the characteristic rates of nuclear magnetic relaxation in proteins. These approaches shed light on a rich and diverse range of motions covering timescales from picoseconds to seconds. Here, we introduce some of the basic concepts upon which these approaches are built and provide a series of illustrations.

Nuclear overhauser spectroscopy of chiral CHD methylene groups.

Nuclear magnetic resonance spectroscopy (NMR) can provide a great deal of information about structure and dynamics of biomolecules. The quality of an NMR structure strongly depends on the number of experimental observables and on their accurate conversion into geometric restraints. When distance restraints are derived from nuclear Overhauser effect spectroscopy (NOESY), stereo-specific assignments of prochiral atoms can contribute significantly to the accuracy of NMR structures of proteins and nucleic acids. Here we introduce a series of NOESY-based pulse sequences that can assist in the assignment of chiral CHD methylene protons in random fractionally deuterated proteins. Partial deuteration suppresses spin-diffusion between the two protons of CH2 groups that normally impedes the distinction of cross-relaxation networks for these two protons in NOESY spectra. Three and four-dimensional spectra allow one to distinguish cross-relaxation pathways involving either of the two methylene protons so that one can obtain stereospecific assignments. In addition, the analysis provides a large number of stereospecific distance restraints. Non-uniform sampling was used to ensure optimal signal resolution in 4D spectra and reduce ambiguities of the assignments. Automatic assignment procedures were modified for efficient and accurate stereospecific assignments during automated structure calculations based on 3D spectra. The protocol was applied to calcium-loaded calbindin D9k. A large number of stereospecific assignments lead to a significant improvement of the accuracy of the structure.