Relaxation Dispersion Methods Research Articles

On the potentially spurious contributions of cosolutes in protein 15N relaxation dispersion measurements

A rigorous quantification of the thermodynamics, kinetic and structural changes can be approached by relaxation dispersion methods, when such motions are in the microsecond to millisecond timescale. Albeit some past efforts, it is still unclear how cosolutes modulate the fitted parameters extracted from relaxation dispersion analyses. Here, we have studied how the systematic measurement of 15N relaxation dispersion on a well-studied enzyme, undergoing two-state chemical exchange, are affected in terms of the populations of the minor state (thermodynamics) and the exchange rates (kinetics) in the presence of different cosolutes, from ions to denaturants. The cosolute-induced changes are modest but can significantly affect protein function. Specifically, exchange rates can be associated to a subtle leverage in the exchange rates. For most canonical residues, i.e. without spurious effects caused by the cosolute, the chemical shift difference (Δω) between both states is essentially unaffected with respect to the expected shift of water resonance. Yet, solvent exposed residues do not always follow this canonical trend. Instead, residue-cosolute interactions are large enough to induce a residue specific shift that ultimately modulates the fitted parameter Δω. Unfortunately, solvent accessibility analysis is not accurate enough to a priori discriminate between canonical and non-canonical residues and a full experimental analysis at varying concentrations of the cosolute is required. This observation aims to act as a word of caution for measurements of relaxation dispersion at a single-solvent condition that contains large amounts of cosolutes.

NMR Relaxation Dispersion Reveals Macrocycle Breathing Dynamics in a Cyclodextrin-based Rotaxane.

A distinctive feature of mechanically interlocked molecules (MIMs) is the relative motion between the mechanically bonded components, and often it is the functional basis for artificial molecular machines and new functional materials. Optimization of machine or materials performance requires knowledge of the underlying atomic-level mechanisms that control the motion. The field of biomolecular NMR spectroscopy has developed a diverse set of pulse schemes that can characterize molecular dynamics over a broad time scale, but these techniques have not yet been used to characterize the motion within MIMs. This study reports the first observation of NMR relaxation dispersion related to MIM motion. The rotary (pirouette) motion of α-cyclodextrin (αCD) wheels was characterized in a complementary pair of rotaxanes with pirouetting switched ON or OFF. 13C and 1H NMR relaxation dispersion measurements reveal previously unknown exchange dynamics for the αCD wheels in the pirouette-ON rotaxane with a rate constant of 2200 s-1 at 298 K and an activation barrier of ΔF‡ = 43 ± 3 kJ/mol. The exchange dynamics disappear in the pirouette-OFF rotaxane, demonstrating their switchable nature. The 13C and 1H sites exhibiting relaxation dispersion suggest that the exchange involves "macrocycle breathing", in which the αCD wheel fluctuates between a contracted or expanded state, the latter enabling diffusive rotary motion about the axle. The substantial insight from these NMR relaxation dispersion methods suggests similar dynamic NMR methods can illuminate the fast time scale (microsecond to millisecond) mechanisms of intercomponent motion in a wide range of MIMs.

Slow ring flips in aromatic cluster of GB1 studied by aromatic 13C relaxation dispersion methods

Ring flips of phenylalanine and tyrosine are a hallmark of protein dynamics. They report on transient breathing motions of proteins. In addition, flip rates also depend on stabilizing interactions in the ground state, like aromatic stacking or cation–π interaction. So far, experimental studies of ring flips have almost exclusively been performed on aromatic rings without stabilizing interactions. Here we investigate ring flip dynamics of Phe and Tyr in the aromatic cluster in GB1. We found that all four residues of the cluster, Y3, F30, Y45 and F52, display slow ring flips. Interestingly, F52, the central residue of the cluster, which makes aromatic contacts with all three others, is flipping significantly faster, while the other rings are flipping with the same rates within margin of error. Determined activation enthalpies and activation volumes of these processes are in the same range of other reported ring flips of single aromatic rings. There is no correlation of the number of aromatic stacking interactions to the activation enthalpy, and no correlation of the ring’s extent of burying to the activation volume. Because of these findings, we speculate that F52 is undergoing concerted ring flips with each of the other rings.

Mechanistic Insights into Microsecond Time-Scale Motion of Solid Proteins Using Complementary 15N and 1H Relaxation Dispersion Techniques.

NMR relaxation dispersion methods provide a holistic way to observe microsecond time-scale protein backbone motion both in solution and in the solid state. Different nuclei (1H and 15N) and different relaxation dispersion techniques (Bloch-McConnell and near-rotary-resonance) give complementary information about the amplitudes and time scales of the conformational dynamics and provide comprehensive insights into the mechanistic details of the structural rearrangements. In this paper, we exemplify the benefits of the combination of various solution- and solid-state relaxation dispersion methods on a microcrystalline protein (α-spectrin SH3 domain), for which we are able to identify and model the functionally relevant conformational rearrangements around the ligand recognition loop occurring on multiple microsecond time scales. The observed loop motions suggest that the SH3 domain exists in a binding-competent conformation in dynamic equilibrium with a sterically impaired ground-state conformation both in solution and in crystalline form. This inherent plasticity between the interconverting macrostates is compatible with a conformational-preselection model and provides new insights into the recognition mechanisms of SH3 domains.

Mapping transiently formed and sparsely populated conformations on a complex energy landscape.

Determining the structures, kinetics, thermodynamics and mechanisms that underlie conformational exchange processes in proteins remains extremely difficult. Only in favourable cases is it possible to provide atomic-level descriptions of sparsely populated and transiently formed alternative conformations. Here we benchmark the ability of enhanced-sampling molecular dynamics simulations to determine the free energy landscape of the L99A cavity mutant of T4 lysozyme. We find that the simulations capture key properties previously measured by NMR relaxation dispersion methods including the structure of a minor conformation, the kinetics and thermodynamics of conformational exchange, and the effect of mutations. We discover a new tunnel that involves the transient exposure towards the solvent of an internal cavity, and show it to be relevant for ligand escape. Together, our results provide a comprehensive view of the structural landscape of a protein, and point forward to studies of conformational exchange in systems that are less characterized experimentally.

Ring flips revisited: (13)C relaxation dispersion measurements of aromatic side chain dynamics and activation barriers in basic pancreatic trypsin inhibitor.

Intramolecular motions of proteins are critical for biological function. Transient structural fluctuations underlie a wide range of processes, including enzyme catalysis, ligand binding to buried sites, and generic protein motions, such as 180° rotation of aromatic side chains in the protein interior, but remain poorly understood. Understanding the dynamics and molecular nature of concerted motions requires characterization of their rates and energy barriers. Here we use recently developed (13)C transverse relaxation dispersion methods to improve our current understanding of aromatic ring flips in basic pancreatic trypsin inhibitor (BPTI). We validate these methods by benchmarking ring-flip rates against the three previously characterized cases in BPTI, namely, Y23, Y35, and F45. Further, we measure conformational exchange for one additional aromatic ring, F22, which can be interpreted in terms of a flip rate of 666 s(-1) at 5 °C. Upon inclusion of our previously reported result that Y21 also flips slowly [Weininger, U., et al. (2013) J. Phys. Chem. B 117, 9241-9247], the (13)C relaxation dispersion experiments thus reveal relatively slow ring-flip rates for five of eight aromatic residues in BPTI. These results are in contrast with previous reports, which have estimated that all rings, except Y23, Y35, and F45, flip with a high rate at ambient temperature. The (13)C relaxation dispersion data result in an updated rank order of ring-flip rates in BPTI, which agrees considerably better with that estimated from a recent 1 ms molecular dynamics trajectory than do previously published NMR data. However, significant quantitative differences remain between experiment and simulation, in that the latter yields flip rates that are in many cases too fast by 1-2 orders of magnitude. By measuring flip rates across a temperature range of 5-65 °C, we determined the activation barriers of ring flips for Y23, Y35, and F45. Y23 and F45 have identical activation parameters, suggesting that the fluctuations of the protein core around these residues are similar in character. Y35 differs from the other two in its apparent activation entropy. These results might be rationalized by the fact that Y23 and F45 are located in the same region of the structure while Y35 is remote from the other two rings. As indicated by our new results for the exceptionally well-characterized protein BPTI, (13)C relaxation dispersion experiments open the possibility of studying ring flips in a range of cases wider than that previously possible.

Chemical exchange in biomacromolecules: Past, present, and future

The perspective reviews quantitative investigations of chemical exchange phenomena in proteins and other biological macromolecules using NMR spectroscopy, particularly relaxation dispersion methods. The emphasis is on techniques and applications that quantify the populations, interconversion kinetics, and structural features of sparsely populated conformational states in equilibrium with a highly populated ground state. Applications to folding, molecular recognition, catalysis, and allostery by proteins and nucleic acids are highlighted.

Transiently populated intermediate functions as a branching point of the FF domain folding pathway

Studies of protein folding and the intermediates that are formed along the folding pathway provide valuable insights into the process by which an unfolded ensemble forms a functional native conformation. However, because intermediates on folding pathways can serve as initiation points of aggregation (implicated in a number of diseases), their characterization assumes an even greater importance. Establishing the role of such intermediates in folding, misfolding, and aggregation remains a major challenge due to their often low populations and short lifetimes. We recently used NMR relaxation dispersion methods and computational techniques to determine an atomic resolution structure of the folding intermediate of a small protein module--the FF domain--with an equilibrium population of 2-3% and a millisecond lifetime, 25 °C. Based on this structure a variant FF domain has been designed in which the native state is selectively destabilized by removing the carboxyl-terminal helix in the native structure to produce a highly populated structural mimic of the intermediate state. Here, we show via solution NMR studies of the designed mimic that the mimic forms distinct conformers corresponding to monomeric and dimeric (K(d) = 0.2 mM) forms of the protein. The conformers exchange on the seconds timescale with a monomer association rate of 1.1 · 10(4) M(-1) s(-1) and with a region responsible for dimerization localized to the amino-terminal residues of the FF domain. This study establishes the FF domain intermediate as a central player in both folding and misfolding pathways and illustrates how incomplete folding can lead to the formation of higher-order structures.

Validation of a Lanthanide Tag for the Analysis of Protein Dynamics by Paramagnetic NMR Spectroscopy

Paramagnetic lanthanide tags potentially can enhance the effects of microsecond to millisecond dynamics in proteins on NMR signals and provide structural information on lowly populated states encoded in the pseudocontact shifts. We have investigated the microsecond to millisecond mobility of a two-point attached lanthanide tag, CLaNP-5, using paramagnetic (1)H CPMG relaxation dispersion methods. CLaNP-5 loaded with Lu(3+), Yb(3+), or Tm(3+) was attached to three sites on the surface of two proteins, pseudoazurin and cytochrome c. The paramagnetic center causes large relaxation dispersion effects for two attachment sites, suggesting that local dynamics of the protein at the attachment site causes mobility of the paramagnetic center. At one site the relaxation dispersions are small and limited to the immediate environment of the tag. It is concluded that paramagnetic relaxation dispersion could represent a sensitive method to probe protein dynamics. However, the selection of a rigid attachment site is of critical importance.

Measurement of Methyl Group Motional Parameters of Invisible, Excited Protein States by NMR Spectroscopy

An understanding of many biological processes can only be achieved through studies of the structure (enthalpy) and motions (entropy) of the key molecules that are involved, including those that are formed only transiently and with low population. These transiently formed, low populated states are invisible to most biophysical techniques but in many cases they can be studied in detail using relaxation dispersion NMR spectroscopy. Relaxation dispersion methodology has recently been described for the measurement of protein backbone excited state chemical shifts as well as bond vector orientations, which form the basis for structural studies of these invisible conformers. It is of interest to extend such studies by quantifying motional parameters of the excited state, providing a more complete description of the energy landscape that drives the biochemical event in question. Herein we describe a relaxation dispersion method for measuring site-specific motional parameters of methyl containing residues in the excited state. The approach is applied to the invisible unfolded state of the G48M Fyn SH3 domain that is in exchange with the folded conformation. Not surprisingly, the degree of disorder is in general higher in the unfolded state than in the folded conformer, although there is some ordering of side-chains in the unfolded state toward the C-terminal region of the domain. The development of the present methodology provides the first step toward characterizing the motional properties of invisible conformers, complementing the structural information that is already available from relaxation dispersion studies.

Partially folded equilibrium intermediate of the villin headpiece HP67 defined by 13C relaxation dispersion

Identification and characterization of ensembles of intermediate states remains an important objective in describing protein folding in atomic detail. The 67-residue villin headpiece, HP67, consists of an N-terminal subdomain (residues 10-42) that transiently unfolds at equilibrium under native-like conditions and a highly stable C-terminal subdomain (residues 43-76). The transition between folded and unfolded states of the N-terminal domain has been characterized previously by (15)N NMR relaxation dispersion measurements (Grey et al. in J Mol Biol 355:1078, 2006). In the present work, (13)C spin relaxation was used to further characterize backbone and hydrophobic core contributions to the unfolding process. Relaxation of (13)C(alpha) spins was measured using the Hahn echo technique at five static magnetic fields (11.7, 14.1, 16.4, 18.8, and 21.1 T) and the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion method at a static magnetic field of 14.1 T. Relaxation of methyl (13)C spins was measured using CPMG relaxation dispersion experiments at static magnetic fields of 14.1 and 18.8 T. Results for (13)C and (15)N spins yielded a consistent model in which the partially unfolded intermediate state of the N-terminal subdomain maintains residual structure for residues near the unprotonated His41 imidazole ring and in the interface between the N- and C-terminal subdomains. In addition, a second faster process was detected that appears to represent local dynamics within the folded state of the molecule and is largely confined to the hydrophobic interface between the N- and C-terminal subdomains.

An Analysis of the Effects of 1HN−1HN Dipolar Couplings on the Measurement of Amide Bond Vector Orientations in Invisible Protein States by Relaxation Dispersion NMR

Marginally and transiently populated conformational states of biomolecules can play important functional roles in biochemical processes. It is of significant interest, therefore, to develop tools for characterizing the structural and dynamical properties of these excited states. One recent development has been the emergence of spin-state-selective relaxation dispersion methods for quantifying dipolar vector orientations in invisible excited-state conformers through measurement of residual dipolar couplings (RDCs). Particularly powerful are 1HN-(15)N RDCs that can be measured with high sensitivity on fractionally aligned, deuterated, uniformly 15N-labeled protein samples. Fractional alignment also produces nonzero 1HN-(1)HN dipolar couplings. These can be problematic for the extraction of robust 1HN-(15)N RDC values, and hence amide bond vector orientations, in cases where the amide proton of interest and a proximal amide proton have small chemical shift differences and a significant 1HN-(1)HN dipolar coupling. Here, we show that while this strong coupling effect leads to aberrant relaxation dispersion profiles, extracted excited-state 1HN-(15)N RDCs are for the most part only marginally affected. Experimental examples of such aberrant profiles are provided, as well as a theoretical consideration of the influence of this strong coupling effect and numerical simulations that assess its impact on extracted parameters.

Protein Self-Association in Solution: The Bovine Pancreatic Trypsin Inhibitor Decamer

We have used magnetic relaxation dispersion to study bovine pancreatic trypsin inhibitor (BPTI) self-association as a function of pH, salt type and concentration, and temperature. The magnetic relaxation dispersion method sensitively detects stable oligomers without being affected by other interactions. We find that BPTI decamers form cooperatively under a wide range of solution conditions with no sign of dimers or other small oligomers. Decamer formation is opposed by electrostatic repulsion among numerous cationic residues confined within a narrow channel. Accordingly, the decamer population increases with increasing pH, as cationic residues are deprotonated, and with increasing salt concentration. The salt effect cannot be described in terms of Debye screening, but involves the ion-specific sequestering of anions within the narrow channel. The lifetime of the BPTI decamer is 101 ± 4 min at 27°C. We propose that the BPTI decamer, with a heparin chain threading the decamer channel, plays a functional role in the mast cell. We also detect a higher oligomer that appears to be a subcritical nucleation cluster of 3–5 decamers. We argue that monomeric crystals form at high pH despite a high decamer population in solution, because the ion pairs that provide the critical decamer-decamer contacts are disrupted at high pH.