Functional protein dynamics on uncharted time scales detected by nanoparticle-assisted NMR spin relaxation.
ABSTRACT: Protein function depends critically on intrinsic internal dynamics, which is manifested in distinct ways, such as loop motions that regulate protein recognition and catalysis. Under physiological conditions, dynamic processes occur on a wide range of time scales from subpicoseconds to seconds. Commonly used NMR spin relaxation in solution provides valuable information on very fast and slow motions but is insensitive to the intermediate nanosecond to microsecond range that exceeds the protein tumbling correlation time. Presently, very little is known about the nature and functional role of these motions. It is demonstrated here how transverse spin relaxation becomes exquisitely sensitive to these motions at atomic resolution when studying proteins in the presence of nanoparticles. Application of this novel cross-disciplinary approach reveals large-scale dynamics of loops involved in functionally critical protein-protein interactions and protein-calcium ion recognition that were previously unobservable.
Project description:Simple and convenient method of protein dynamics evaluation from the insufficient experimental 15N relaxation data is presented basing on the ratios, products, and differences of longitudinal and transverse 15N relaxation rates obtained at a single magnetic field. Firstly, the proposed approach allows evaluating overall tumbling correlation time (nanosecond time scale). Next, local parameters of the model-free approach characterizing local mobility of backbone amide N-H vectors on two different time scales, S2 and R ex , can be elucidated. The generalized order parameter, S2, describes motions on the time scale faster than the overall tumbling correlation time (pico- to nanoseconds), while the chemical exchange term, R ex , identifies processes slower than the overall tumbling correlation time (micro- to milliseconds). Advantages and disadvantages of different methods of data handling are thoroughly discussed.
Project description:The HIV-1 transactivation response element (TAR) RNA binds a variety of proteins and is a target for developing anti-HIV therapies. TAR has two primary binding sites: a UCU bulge and a CUGGGA apical loop. We used NMR residual dipolar couplings, carbon spin relaxation (R(1) and R(2)), and relaxation dispersion (R(1rho)) in conjunction with molecular dynamics and mutagenesis to characterize the dynamics of the TAR apical loop and investigate previously proposed long-range interactions with the distant bulge. Replacement of the wild-type apical loop with a UUCG loop did not significantly affect the structural dynamics at the bulge, indicating that the apical loop and the bulge act largely as independent dynamical recognition centers. The apical loop undergoes complex dynamics at multiple timescales that are likely important for adaptive recognition: U31 and G33 undergo limited motions, G32 is highly flexible at picosecond-nanosecond timescales, and G34 and C30 form a dynamic Watson-Crick basepair in which G34 and A35 undergo a slow (approximately 30 mus) likely concerted looping in and out motion, with A35 also undergoing large amplitude motions at picosecond-nanosecond timescales. Our study highlights the power of combining NMR, molecular dynamics, and mutagenesis in characterizing RNA dynamics.
Project description:Many RNAs undergo large conformational changes in response to the binding of proteins and small molecules. However, when RNA functional dynamics occur in the nanosecond-microsecond time scale, they become invisible to traditional solution NMR relaxation methods. Residual dipolar coupling methods have revealed the presence of extensive nanosecond-microsecond domain motions in HIV-1 TAR RNA, but this technique lacks information on the rates of motions. We have used solid-state deuterium NMR to quantitatively describe trajectories of key residues in TAR by exploiting the sensitivity of this technique to motions that occur in the nanosecond-microsecond regime. Deuterium line shape and relaxation data were used to model motions of residues within the TAR binding interface. The resulting motional models indicate two functionally essential bases within the single-stranded bulge sample both the free and Tat-bound conformations on the microsecond time scale in the complete absence of the protein. Thus, our results strongly support a conformational capture mechanism for recognition: the protein does not induce a new RNA structure, but instead captures an already-populated conformation.
Project description:Transverse relaxation rate measurements in magic-angle spinning solid-state nuclear magnetic resonance provide information about molecular motions occurring on nanosecond-to-millisecond (ns-ms) time scales. The measurement of heteronuclear ((13)C, (15)N) relaxation rate constants in the presence of a spin-lock radiofrequency field (R1? relaxation) provides access to such motions, and an increasing number of studies involving R1? relaxation in proteins have been reported. However, two factors that influence the observed relaxation rate constants have so far been neglected, namely, (1) the role of CSA/dipolar cross-correlated relaxation (CCR) and (2) the impact of fast proton spin flips (i.e., proton spin diffusion and relaxation). We show that CSA/D CCR in R1? experiments is measurable and that the CCR rate constant depends on ns-ms motions; it can thus provide insight into dynamics. We find that proton spin diffusion attenuates this CCR due to its decoupling effect on the doublet components. For measurements of dynamics, the use of R1? rate constants has practical advantages over the use of CCR rates, and this article reveals factors that have so far been disregarded and which are important for accurate measurements and interpretation.
Project description:Sequence-specific DNA flexibility plays a key role in a variety of cellular interactions that are critical for gene packaging, expression, and regulation, yet few studies have experimentally explored the sequence dependence of DNA dynamics that occur on biologically relevant time scales. Here, we use nuclear magnetic resonance (NMR) carbon spin relaxation combined with molecular dynamics (MD) simulations to examine the picosecond to nanosecond dynamics in a variety of dinucleotide steps as well as in varying length homopolymeric A(n)·T(n) repeats (A(n)-tracts, where n = 2, 4, or 6) that exhibit unusual structural and mechanical properties. We extend the NMR spin relaxation time scale sensitivity deeper into the nanosecond regime by using glycerol and a longer DNA duplex to slow overall tumbling. Our studies reveal a structurally unique A-tract core (for n > 3) that is uniformly rigid, flanked by junction steps that show increasing sugar flexibility with A-tract length. High sugar mobility is observed at pyrimidine residues at the A-tract junctions, which is encoded at the dinucleotide level (CA, TG, and CG steps) and increases with A-tract length. The MD simulations reproduce many of these trends, particularly the overall rigidity of A-tract base and sugar sites, and suggest that the sugar-backbone dynamics could involve transitions in sugar pucker and phosphate backbone BI ? BII equilibria. Our results reinforce an emerging view that sequence-specific DNA flexibility can be imprinted in dynamics occurring deep within the nanosecond time regime that is difficult to characterize experimentally at the atomic level. Such large-amplitude sequence-dependent backbone fluctuations might flag the genome for specific DNA recognition.
Project description:The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR (15)N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R1 relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.
Project description:Solid-state NMR spectroscopy can be used to probe internal protein dynamics in the absence of the overall molecular tumbling. In this study, we report (15)N backbone dynamics in differentially enriched 1-73(U-(13)C,(15)N)/74-108(U-(15)N) reassembled thioredoxin on multiple time scales using a series of 2D and 3D MAS NMR experiments probing the backbone amide (15)N longitudinal relaxation, (1)H-(15)N dipolar order parameters, (15)N chemical shift anisotropy (CSA), and signal intensities in the temperature-dependent and (1)H T(2)'-filtered NCA experiments. The spin-lattice relaxation rates R(1) (R(1) = 1/T(1)) were observed in the range from 0.012 to 0.64 s(-1), indicating large site-to-site variations in dynamics on pico- to nanosecond time scales. The (1)H-(15)N dipolar order parameters, <S>, and (15)N CSA anisotropies, delta(sigma), reveal the backbone mobilities in reassembled thioredoxin, as reflected in the average <S> = 0.89 +/- 0.06 and delta(sigma) = 92.3 +/- 5.2 ppm, respectively. From the aggregate of experimental data from different dynamics methods, some degree of correlation between the motions on the different time scales has been suggested. Analysis of the dynamics parameters derived from these solid-state NMR experiments indicates higher mobilities for the residues constituting irregular secondary structure elements than for those located in the alpha-helices and beta-sheets, with no apparent systematic differences in dynamics between the alpha-helical and beta-sheet residues. Remarkably, the dipolar order parameters derived from the solid-state NMR measurements and the corresponding solution NMR generalized order parameters display similar qualitative trends as a function of the residue number. The comparison of the solid-state dynamics parameters to the crystallographic B-factors has identified the contribution of static disorder to the B-factors. The combination of longitudinal relaxation, dipolar order parameter, and CSA line shape analyses employed in this study provides snapshots of dynamics and a new insight on the correlation of these motions on multiple time scales.
Project description:Understanding the molecular determinants underlying protein function requires the characterization of both structure and dynamics at atomic resolution. Nuclear relaxation rates allow a precise characterization of protein dynamics at the Larmor frequencies of spins. This usually limits the sampling of motions to a narrow range of frequencies corresponding to high magnetic fields. At lower fields one cannot achieve sufficient sensitivity and resolution in NMR. Here, we use a fast shuttle device where the polarization builds up and the signals are detected at high field, while longitudinal relaxation takes place at low fields 0.5 < B0 < 14.1 T. The sample is propelled over a distance up to 50 cm by a blowgun-like system in about 50 ms. The analysis of nitrogen-15 relaxation in the protein ubiquitin over such a wide range of magnetic fields offers unprecedented insights into molecular dynamics. Some key regions of the protein feature structural fluctuations on nanosecond time scales, which have so far been overlooked in high-field relaxation studies. Nanosecond motions in proteins may have been underestimated by traditional high-field approaches, and slower supra-?(c) motions that have no effect on relaxation may have been overestimated. High-resolution relaxometry thus opens the way to a quantitative characterization of nanosecond motions in proteins.
Project description:According to NMR chemical shift data, the ensemble of ubiquitin is a mixture of "open" and "closed" conformations at rapid equilibrium. Pressure perturbations provide the means to study the transition between the two conformers by imposing an additional constraint on the system's partial molar volume. Here we use nanosecond-timescale molecular dynamics simulations to characterize the network of correlated motions accessible to the conformers at low- and high-pressure conditions. Using the isotropic reorientational eigenmode dynamics formalism to analyze our simulation trajectories, we reproduce NMR relaxation data without fitting any parameters of our model. Comparative analysis of our results suggests that the two conformations behave very differently. The dynamics of the "closed" conformation are almost unaffected by pressure and are dominated by large-amplitude correlated motions of residues 23-34 in the extended alpha-helix. The "open" conformation under conditions of normal pressure displays increased mobility, focused on the loop residues 17-20, 46-55, and 58-59 at the bottom of the core of the structure, as well as the C-terminal residues 69-76, that directly participate in key protein-protein interactions. For the same conformation, a pressure increase induces a loss of separability between molecular tumbling and internal dynamics, while motions between different backbone sites become uncorrelated.
Project description:Electron paramagnetic resonance (EPR) at 236.6 and 9.5 GHz probed the tumbling of nitroxide spin probes in the lower stem, in the upper loop, and near the bulge of mini c TAR DNA. High-frequency 236.6 GHz EPR, not previously applied to spin-labeled oligonucleotides, was notably sensitive to fast, anisotropic, hindered local rotational motion of the spin probe, occurring approximately about the NO nitroxide axis. Labels attached to the 2'-aminocytidine sugar in the mini c TAR DNA showed such anisotropic motion, which was faster in the lower stem, a region previously thought to be partially melted. More flexible labels attached to phosphorothioates at the end of the lower stem tumbled isotropically in mini c TAR DNA, mini TAR RNA, and ?(3) RNA, but at 5 °C, the motion became more anisotropic for the labeled RNAs, implying more order within the RNA lower stems. As observed by 9.5 GHz EPR, the slowing of nanosecond motions of large segments of the oligonucleotide was enhanced by increasing the ratio of the nucleocapsid protein NCp7 to mini c TAR DNA from 0 to 2. The slowing was most significant at labels in the loop and near the bulge. At a 4:1 ratio of NCp7 to mini c TAR DNA, all labels reported tumbling times of >5 ns, indicating a condensation of NCp7 and TAR DNA. At the 4:1 ratio, pulse dipolar EPR spectroscopy of bilabels attached near the 3' and 5' termini showed evidence of an NCp7-induced increase in the 3'-5' end-to-end distance distribution and a partially melted stem.