R1ρ Relaxation Dispersion Research Articles

Microsecond Timescale Conformational Dynamics of a Small-Molecule Ligand within the Active Site of a Protein.

The possible internal dynamics of non-isotope-labeled small-molecule ligands inside a target protein is inherently difficult to capture. Whereas high crystallographic temperature factors can denote either static disorder or motion, even moieties with very low B-factors can be subject to vivid motion between symmetry-related sites. Here we report the experimental identification of internal μs timescale dynamics of a high-affinity, natural-abundance ligand tightly bound to the enzyme human carbonic anhydrase II (hCAII) even within a crystalline lattice. The rotamer jumps of the ligand's benzene group manifest themselves both, in solution and fast magic-angle spinning solid-state NMR 1 H R1ρ relaxation dispersion, for which we obtain further mechanistic insights from molecular-dynamics (MD) simulations. The experimental confirmation of rotameric jumps in bound ligands within proteins in solution or the crystalline state may improve understanding of host-guest interactions in biology and supra-molecular chemistry and may facilitate medicinal chemistry for future drug campaigns.

Konformationeller Austausch eines Liganden in der Bindetasche eines Proteins auf der Mikrosekundenzeitskala

AbstractDie experimentelle Erfassung möglicher interner Dynamik eines Liganden ohne Isotopenmarkierung im proteingebundenen Zustand ist grundsätzlich schwierig. Hohe Temperaturfaktoren aus Kristallstrukturen können statische Heterogenität nicht von Bewegung unterscheiden, zudem können für symmetrische Bewegungen selbst bei niedrigsten B‐Faktoren schnelle Bewegungen vorhanden sein. Hier zeigen wir die sowohl in Lösung als auch im Kristall experimentell beobachtete interne μs‐Dynamik eines nicht‐isotopenmarkierten Liganden, während dieser mit hoher Affinität an das Enzym humane Carboanhydrase II (hCAII) gebunden ist. Der sprunghafte Wechsel zwischen den Rotameren der Benzolgruppe des Liganden verursacht sowohl in Lösungs‐ als auch Festkörper NMR mit schneller Rotation um den magischen Winkel 1H R1ρ Relaxationsdispersion, für die mittels Molecular Dynamics (MD)‐Simulationen mechanistische Einblicke erhalten werden. Die experimentelle Erfassung von konformationellem Austausch in gebundenen Liganden in Lösung und im Festkörper könnte einem besseren Verständnis von Wirt‐Gast‐Interaktionen in Biologie und supramolekularer und medizinischer Chemie dienen.

Site-based description of R1ρ relaxation in local reference frames

Nuclear magnetic spin relaxation in the presence of an applied radiofrequency field depends critically on chemical exchange processes that transfer nuclear spins between chemical or conformational environments with distinct resonance frequencies. Characterization of chemical exchange processes in R1ρ relaxation dispersion, CEST, and DEST experiments provides powerful insights into chemical and conformational kinetics of biological macromolecules. The present work reformulates expressions for magnetization evolution and the R1ρ relaxation rate constant by focussing on the orientations of the tilted rotating frames of reference for magnetization components in different sites, by treating the spin-locking field strength as a perturbation to free-precession evolution, and by applying the Homotopy Analysis and Laplace transform methods to approximate solutions to the Bloch-McConnell equations. The results provide an expression for R1ρ that is invariant to the topology of the kinetic scheme, an approximate equation for evolution of spin-locked z-magnetization, and an approach for effective simplification of chemical exchange topologies for 2- and N-site chemical exchange processes. The theoretical approach also provides an accurate approximation for relaxation during a constant-amplitude radiofrequency field in the absence of exchange.

NMR Methods to Study the Dynamics of SH2 Domain-Phosphopeptide Complexes.

Nuclear magnetic resonance (NMR) spectroscopy is the method of choice for studying the dynamics of biological macromolecules in solution. By exploiting the intricate interplay between the effects of protein motion (both overall rotational diffusion and internal mobility) and nuclear spin relaxation, NMR allows molecular motion to be probed at atomic resolution over a wide range of timescales, including picosecond (bond vibrations and methyl-group rotations), nanosecond (loop motions and rotational diffusion), and microsecond-millisecond (ligand binding, allostery). In this chapter, we describe different NMR pulse schemes (R1, R1ρ, heteronuclear NOE, and CPMG relaxation dispersion) to characterize the dynamics of SH2 domains. As an example, we use the N-SH2 domain of protein tyrosine phosphatase SHP2 in complex with two phosphopeptides derived from immune checkpoint receptor PD-1 (ITIM and ITSM).

Mutate-and-chemical-shift-fingerprint (MCSF) to characterize excited states in RNA using NMR spectroscopy.

It is important to understand the dynamics and higher energy structures of RNA, called excited states, to achieve better understanding of RNA function. R1ρ relaxation dispersion NMR spectroscopy (RD) determines chemical shift differences between the most stable, ground state and the short-lived, low-populated excited states. We describe a procedure for deducing the excited state structure from these chemical shift differences using the mutate-and-chemical-shift-fingerprint (MCSF) method, which requires ~2-6 weeks and moderate understanding of NMR and RNA structure. We recently applied the MCSF methodology to elucidate the excited state of microRNA 34a targeting the SIRT1 mRNA and use this example to demonstrate the analysis. The protocol comprises the following steps: (i) determination of the secondary structure of the excited state from RD chemical shift data, (ii) design of trapped excited state RNA, (iii) validation of the excited state structure by NMR, and (iv) MCSF analysis comparing the chemical shifts of the trapped excited state with the RD-derived chemical shift differences. MCSF enables observation of the short-lived RNA structures, which can be functionally and structurally characterized by entrapment.

Practical Aspects of Sample Preparation and Setup of <sup>1</sup>H <em>R<sub>1ρ</sub></em> Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1ρ relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing. The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1ρ experiments with both isotopically labeled and unlabeled RNA samples.

Practical Aspects of Sample Preparation and Setup of <sup>1</sup>H <em>R<sub>1ρ</sub></em> Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1ρ relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing. The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1ρ experiments with both isotopically labeled and unlabeled RNA samples.

Single Base-Pair Conformational Switch Modulates miR-34a Targeting of Sirt1 mRNA

MicroRNAs (miRNAs) regulate numerous messenger RNAs levels and translation, via binding to the protein Argonaute (Ago). The base pairing between the miRNA and mRNA in the seed region governs target selection and repression efficiency. Biochemical studies and X-ray crystallography structures elucidated the role of Ago and miRNA in this complex, however, structural information on the miRNA-mRNA duplex and its dynamics are still missing. We employ Nuclear Magnetic Resonance R1ρ Relaxation Dispersion and molecular simulations to study the structure and dynamics of miR-34a binding to mRNA of Sirt1, regulating p53 activity. We reveal a switching mode based on a single base pair rearrangement in the miRNA-mRNA duplex, elongating the 6/7mer seed to an 8mer seed. The base pair switch correlates with changing in overall structure of these conformers, which, in hAgo2, indicates two distinct binding modes and the stabilization of the minor conformer exhibit enhanced target repression in cells, shown by luciferase reporter assay. This change in activity indicates that the conformational switch in the miRNA-mRNA duplex can push the complex from an initial “screening” state to an “active” state. Our observations tie together current understanding of the step-wise miRNA target recognition process and shed light on the role of miRNA binding to mRNA beyond the seed.

Structural dynamics of double-stranded DNA with epigenome modification.

Modification of cytosine plays an important role in epigenetic regulation of gene expression and genome stability. Cytosine is converted to 5-methylcytosine (5mC) by DNA methyltransferase; in turn, 5mC may be oxidized to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation enzyme. The structural flexibility of DNA is known to affect the binding of proteins to methylated DNA. Here, we have carried out a semi-quantitative analysis of the dynamics of double-stranded DNA (dsDNA) containing various epigenetic modifications by combining data from imino 1H exchange and imino 1H R1ρ relaxation dispersion NMR experiments in a complementary way. Using this approach, we characterized the base-opening (kopen) and base-closing (kclose) rates, facilitating a comparison of the base-opening and -closing process of dsDNA containing cytosine in different states of epigenetic modification. A particularly striking result is the increase in the kopen rate of hemi-methylated dsDNA 5mC/C relative to unmodified or fully methylated dsDNA, indicating that the Watson–Crick base pairs undergo selective destabilization in 5mC/C. Collectively, our findings imply that the epigenetic modulation of cytosine dynamics in dsDNA mediates destabilization of the GC Watson–Crick base pair to allow base-flipping in living cells.

RING NMR dynamics: software for analysis of multiple NMR relaxation experiments

Molecular motions are fundamental to the existence of life, and NMR spectroscopy remains one of the most useful and powerful methods to measure their rates and molecular characteristics. Multiple experimental methods are available for measuring the NMR relaxation properties and these can require different methods for extracting model parameters. We present here a new software application, RING NMR Dynamics, that is designed to support analysis of multiple relaxation types. The initial release of RING NMR Dynamics supports the analysis of exponential decay experiments such as T1 and T2, as well as CEST and R2 and R1ρ relaxation dispersion. The software runs on multiple operating systems in both a command line mode and a user-friendly GUI that allows visualizing and simulating relaxation data. Interaction with another program, NMRFx Analyst, allows drilling down from the derived relaxation parameters to the raw spectral data.

NMR Chemical Exchange Measurements Reveal That N6-Methyladenosine Slows RNA Annealing.

N6-Methyladenosine (m6A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. While m6A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA-RNA interactions by altering RNA structure mainly by destabilizing base pairing. Little is known about how m6A and other epitranscriptomic modifications might affect the kinetic rates of RNA folding and other conformational transitions that are also important for cellular activity. Here, we used NMR R1ρ relaxation dispersion and chemical exchange saturation transfer to noninvasively and site-specifically measure nucleic acid hybridization kinetics. The methodology was validated on two DNA duplexes and then applied to examine how a single m6A alters the hybridization kinetics in two RNA duplexes. The results show that m6A minimally impacts the rate constant for duplex dissociation, changing koff by ∼1-fold but significantly slows the rate of duplex annealing, decreasing kon by ∼7-fold. A reduction in the annealing rate was observed robustly for two different sequence contexts at different temperatures, both in the presence and absence of Mg2+. We propose that rotation of the N6-methyl group from the preferred syn conformation in the unpaired nucleotide to the energetically disfavored anti conformation required for Watson-Crick pairing is responsible for the reduced annealing rate. The results help explain why in mRNA m6A slows down tRNA selection and more generally suggest that m6A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.

Direct evidence for (G)O6···H2-N4(C)+ hydrogen bonding in transient G(syn)-C+ and G(syn)-m5C+ Hoogsteen base pairs in duplex DNA from cytosine amino nitrogen off-resonance R1ρ relaxation dispersion measurements.

NMR relaxation dispersion studies have shown that Watson-Crick G-C and A-T base pairs in duplex DNA exist in dynamic equilibrium with their Hoogsteen counterparts. Hoogsteen base pairs form through concurrent rotation of the purine base about the glycosidic bond from an anti to a syn conformation and constriction of the C1'-C1' distance across the base pair by ∼2 Å to allow Hoogsteen type hydrogen bonding. Owing to their unique structure, Hoogsteen base pairs can play important roles in DNA recognition, the accommodation, recognition, and repair of DNA damage, and in DNA replication. NMR relaxation dispersion experiments targeting imino nitrogen and protonated base and sugar carbons have provided insights into many structural features of transient Hoogsteen base pairs, including one of two predicted hydrogen bonds involving (G)N7···H-N3(C)+ and (A)N7···H-N3(T). Here, through measurement of cytosine amino (N4) R1ρ relaxation dispersion, we provide direct evidence for the second (G)O6···H2-N4(C)+ hydrogen bond in G(syn)-C+ transient Hoogsteen base pairs. The utility of cytosine N4 R1ρ relaxation dispersion as a new sensitive probe of transient Hoogsteen base pairs, and cytosine dynamics in general, is further demonstrated by measuring G(syn)-C+ Hoogsteen exchange near neutral pH and in the context of the naturally occurring DNA modification 5-methyl cytosine (m5C), in DNA samples prepared using chemical synthesis and a 15N labeled m5C phosphoramidite.

Quantifying Microsecond Exchange in Large Protein Complexes with Accelerated Relaxation Dispersion Experiments in the Solid State

Solid state NMR is a powerful method to obtain information on the structure and dynamics of protein complexes that, due to solubility and size limitations, cannot be achieved by other methods. Here, we present an approach that allows the quantification of microsecond conformational exchange in large protein complexes by using a paramagnetic agent to accelerate 15N R1ρ relaxation dispersion measurements and overcome sensitivity limitations. The method is validated on crystalline GB1 and then applied to a >300 kDa precipitated complex of GB1 with full length human immunoglobulin G (IgG). The addition of a paramagnetic agent increased the signal to noise ratio per time unit by a factor of 5, which allowed full relaxation dispersion curves to be recorded on a sample containing less than 50 μg of labelled material in 5 and 10 days on 850 and 700 MHz spectrometers, respectively. We discover a similar exchange process across the β-sheet in GB1 in crystals and in complex with IgG. However, the slow motion observed for a number of residues in the α-helix of crystalline GB1 is not detected in the complex.

Conformational Dynamics in the Core of Human Y145Stop Prion Protein Amyloid Probed by Relaxation Dispersion NMR.

Microsecond to millisecond timescale backbone dynamics of the amyloid core residues in Y145Stop human prion protein (PrP) fibrils were investigated by using 15 N rotating frame (R1ρ ) relaxation dispersion solid-state nuclear magnetic resonance spectroscopy over a wide range of spin-lock fields. Numerical simulations enabled the experimental relaxation dispersion profiles for most of the fibril core residues to be modelled by using a two-state exchange process with a common exchange rate of 1000 s-1 , corresponding to protein backbone motion on the timescale of 1 ms, and an excited-state population of 2 %. We also found that the relaxation dispersion profiles for several amino acids positioned near the edges of the most structured regions of the amyloid core were better modelled by assuming somewhat higher excited-state populations (∼5-15 %) and faster exchange rate constants, corresponding to protein backbone motions on the timescale of ∼100-300 μs. The slow backbone dynamics of the core residues were evaluated in the context of the structural model of human Y145Stop PrP amyloid.

Resolving biomolecular motion and interactions by R2 and R1ρ relaxation dispersion NMR.

Among the tools of structural biology, NMR spectroscopy is unique in that it not only derives a static three-dimensional structure, but also provides an atomic-level description of the local fluctuations and global dynamics around this static structure. A battery of NMR experiments is now available to probe the motions of proteins and nucleic acids over the whole biologically relevant timescale from picoseconds to hours. Here we focus on one of these methods, relaxation dispersion, which resolves dynamics on the micro- to millisecond timescale. Key biological processes that occur on this timescale include enzymatic catalysis, ligand binding, and local folding. In other words, relaxation-dispersion-resolved dynamics are often closely related to the function of the molecule and therefore highly interesting to the structural biochemist. With an astounding sensitivity of ∼0.5%, the method detects low-population excited states that are invisible to any other biophysical method. The kinetics of the exchange between the ground state and excited states are quantified in the form of the underlying exchange rate, while structural information about the invisible excited state is obtained in the form of its chemical shift. Lastly, the population of the excited state can be derived. This diversity in the information that can be obtained makes relaxation dispersion an excellent method to study the detailed mechanisms of conformational transitions and molecular interactions. Here we describe the two branches of relaxation dispersion, R2 and R1ρ, discussing their applicability, similarities, and differences, as well as recent developments in pulse sequence design and data processing.

Overview of Relaxation Dispersion NMR Spectroscopy to Study Protein Dynamics and Protein-Ligand Interactions.

Proteins and nucleic acids are central to all biological processes. NMR spectroscopy has proven to be excellent for studying the dynamics of these macromolecules over various timescales. Relaxation rates and heteronuclear nuclear Overhauser-effect values can resolve motion on pico- to nanosecond timescales, residual dipolar couplings provide information on submicro- to millisecond timescales, and even slower dynamics over seconds to hours can be resolved by hydrogen-exchange experiments. Relaxation dispersion experiments are especially valuable because they resolve motion on micro- to millisecond timescales, encompassing biomolecular motions associated with ligand binding, enzymatic catalysis, and domain-domain opening. These experiments provide structural, kinetic, and thermodynamic information on "invisible" excited conformational states. Relaxation dispersion can be applied not only to single biomolecules but also to protein-ligand complexes to study the kinetics and thermodynamics of association and dissociation. We review recent developments in relaxation dispersion methodology, outline the R1ρ relaxation dispersion experiment, and discuss application to biomolecular interactions. © 2018 by John Wiley & Sons, Inc.

Probing Conformational Exchange in Weakly Interacting, Slowly Exchanging Protein Systems via Off-Resonance R1ρ Experiments: Application to Studies of Protein Phase Separation.

R1ρ relaxation dispersion experiments are increasingly used in studies of protein dynamics on the micro- to millisecond time scale. Traditional R1ρ relaxation dispersion approaches are typically predicated on changes in chemical shifts between corresponding probe spins, ΔωGE, in the interconverting states. Here, we present a new application of off-resonance 15N R1ρ relaxation dispersion that enables the quantification of slow exchange processes even in the limit where ΔωGE = 0 so long as the spins in the exchanging states have different intrinsic transverse relaxation rates (ΔR2 = R2,E - R2,G ≠ 0). In this limit, the dispersion profiles become inverted relative to those measured in the case where ΔωGE ≠ 0, ΔR2 = 0. The theoretical background to understand this effect is presented, along with a simplified exchange matrix that is valid in the limits that are germane here. An application to the study of the dynamics of the germ granule protein Ddx4 in a highly concentrated phase-separated state is described. Notably, exchange-based dispersion profiles can be obtained despite the fact that ΔωGE ≈ 0 and ΔR2 is small, ∼20-30 s-1. Our results are consistent with the formation of a significantly populated excited conformational state that displays increased contacts between adjacent protein molecules relative to the major conformer in solution, leading to a decrease in overall motion of the protein backbone. A complete set of exchange parameters is obtained from analysis of a single set of 15N off-resonance R1ρ measurements recorded at a single static magnetic field and with a single spin-lock radio frequency field strength. This new approach holds promise for studies of weakly interacting systems, especially those involving intrinsically disordered proteins that form phase-separated organelles, where little change to chemical shifts between interconverting states would be expected, but where finite ΔR2 values are observed.

Microsecond Timescale Protein Dynamics: a Combined Solid-State NMR Approach.

Conformational exchange in proteins is a major determinant in protein functionality. In particular, the μs-ms timescale is associated with enzymatic activity and interactions between biological molecules. We show here that a comprehensive data set of R1ρ relaxation dispersion profiles employing multiple effective fields and tilt angles can be easily obtained in perdeuterated, partly back-exchanged proteins at fast magic-angle spinning and further complemented with chemical-exchange saturation transfer NMR experiments. The approach exploits complementary sources of information and enables the extraction of multiple exchange parameters for μs-ms timescale conformational exchange, most notably including the sign of the chemical shift differences between the ground and excited states.

Protein conformational dynamics studied by 15N and 1H R1ρ relaxation dispersion: Application to wild-type and G53A ubiquitin crystals

Solid-state NMR spectroscopy can provide site-resolved information about protein dynamics over many time scales. Here we combine protein deuteration, fast magic-angle spinning (~45–60kHz) and proton detection to study dynamics of ubiquitin in microcrystals, and in particular a mutant in a region that undergoes microsecond motions in a β-turn region in the wild-type protein. We use 15N R1ρ relaxation measurements as a function of the radio-frequency (RF) field strength, i.e. relaxation dispersion, to probe how the G53A mutation alters these dynamics. We report a population-inversion of conformational states: the conformation that in the wild-type protein is populated only sparsely becomes the predominant state. We furthermore explore the potential to use amide-1H R1ρ relaxation to obtain insight into dynamics. We show that while quantitative interpretation of 1H relaxation remains beyond reach under the experimental conditions, due to coherent contributions to decay, one may extract qualitative information about flexibility.

Capturing Excited States in the Fast‐Intermediate Exchange Limit in Biological Systems Using 1H NMR Spectroscopy

AbstractChanges in molecular structure are essential for the function of biomolecules. Characterization of these structural fluctuations can illuminate alternative states and help in correlating structure to function. NMR relaxation dispersion (RD) is currently the only method for detecting these alternative, high‐energy states. In this study, we present a versatile 1H R1ρ RD experiment that not only extends the exchange timescales at least three times beyond the rate limits of 13C/15N R1ρ and ten times for CPMG experiments, but also makes use of easily accessible probes, thus allowing a general description of biologically important excited states. This technique can be used to extract chemical shifts for the structural characterization of excited states and to elucidate complex excited states.