Triple-resonance Spectroscopy Research Articles

Robust automated backbone triple resonance NMR assignments of proteins using Bayesian-based simulated annealing

Assignment of resonances of nuclear magnetic resonance (NMR) spectra to specific atoms within a protein remains a labor-intensive and challenging task. Automation of the assignment process often remains a bottleneck in the exploitation of solution NMR spectroscopy for the study of protein structure-dynamics-function relationships. We present an approach to the assignment of backbone triple resonance spectra of proteins. A Bayesian statistical analysis of predicted and observed chemical shifts is used in conjunction with inter-spin connectivities provided by triple resonance spectroscopy to calculate a pseudo-energy potential that drives a simulated annealing search for the most optimal set of resonance assignments. Termed Bayesian Assisted Assignments by Simulated Annealing (BARASA), a C++ program implementation is tested against systems ranging in size to over 450 amino acids including examples of intrinsically disordered proteins. BARASA is fast, robust, accommodates incomplete and incorrect information, and outperforms current algorithms – especially in cases of sparse data and is sufficiently fast to allow for real-time evaluation during data acquisition.

Spin Polarization Reveals the Coordination Geometry of the [FeFe] Hydrogenase Active Site in Its CO-Inhibited State.

The active site of [FeFe] hydrogenase features a binuclear iron cofactor Fe2ADT(CO)3(CN)2, where ADT represents the bridging ligand aza-propane-dithiolate. The terminal diatomic ligands all coordinate in a basal configuration, and one CO bridges the two irons leaving an open coordination site at which the hydrogen species and the competitive inhibitor CO bind. Externally supplied CO is expected to coordinate in an apical configuration. However, an alternative configuration has been proposed in which, due to ligand rotation, the CN– bound to the distal Fe becomes apical. Using selective 13C isotope labeling of the CN– and COext ligands in combination with pulsed 13C electron–nuclear–nuclear triple resonance spectroscopy, spin polarization effects are revealed that, according to density functional theory calculations, are consistent with only the “unrotated” apical COext configuration.

The evolution of solution state NMR pulse sequences through the 'eyes' of triple-resonance spectroscopy.

Careful pulse sequence design and optimization is critical to the success of a given NMR experiment. Over the past several decades the level of sophistication of NMR pulse sequences has increased tremendously, leading to large spectral sensitivity and resolution improvements, to data sets with far fewer artifacts, and to much more rapid acquisition times, opening up a wide range of applications. Here I briefly highlight how pulse sequence 'engineering' has evolved, focusing on liquid state NMR, and, in particular, on the HNCA-class of triple-resonance experiment. In many respects, the evolution of triple-resonance NMR mirrors the evolution of solution state NMR experiments in general, with 'tricks' that first appeared in triple-resonance pulse sequences or that were motivated by them now incorporated into a broad range of experiments.

NMR characterization of a 72 kDa transcription factor using differential isotopic labeling.

NF-κB is a major transcription factor that mediates a number of cellular signaling pathways. Crystal structure analysis gives an incomplete picture of the behavior of the protein, particularly in the free state; free monomers or dimers of NF-κB have never been crystallized. NMR analysis gives insights into the structure and dynamics of the protein in solution, but a necessary first step is the assignment of resonances. The size of the heterodimer of the Rel homology regions of the NF-κB monomers p65 and p50 (72 kDa) prohibits the straightforward use of triple-resonance spectroscopy to obtain the assignments. However, the dynamic nature of the free heterodimer, in particular the independence of the DNA-binding and dimerization domains of each monomer, allows the assignments made on differentially labeled smaller domains to be mapped successfully onto the spectrum of the larger full-length RHR. Problematic areas such as the p65 nuclear localization sequence, which is disordered in the free protein, can be approached by residue-specific labeling and comparison with previously-published spectra of a short peptide with the same sequence. Overall, this NMR analysis of NF-κB has given valuable insights into the highly dynamic nature of the free state, which is likely to play an important role in the functional cycle of NF-κB in the cell.

Dissociation threshold of H218O: Validating ab initio calculations by state-selective triple-resonance spectroscopy

We employ state-selective triple-resonance vibrational overtone spectroscopy for a direct measurement of the lowest dissociation energy, D0, in the H218O molecule. The measured value of 41154.22±0.25cm−1 is in an excellent agreement with that, predicted by ab initio theoretical calculations. This result validates the computations, adding more confidence to what this theory can predict for water. In addition, we have measured the required for this experiment energy levels of H218O in the region near 27000cm−1, as well as the laser-induced fluorescence (LIF) spectrum of the 18OH isotopologue, for which near 60 electronic transitions have been assigned.

H(C)Ag: a triple resonance NMR experiment for (109) Ag detection in labile silver-carbene complexes.

In silver complexes, indirect detection of (109) Ag resonances via (1) H,(109) Ag-HMQC frequently suffers from small or absent JHAg couplings or rapid ligand dissociation. In these cases, it would be favourable to employ H(X)Ag triple resonance spectroscopy that uses the large one-bond JXAg coupling (where the donor atom of the ligand X is the relay nucleus). We have applied an HMQC-based version of the H(C)Ag experiment to a labile silver-NHC complex (NHC=N-heterocyclic carbene) at natural (13) C isotopic abundance and variable temperature. In agreement with simulations, H(C)Ag detection became superior to (1) H,(109) Ag-HMQC detection above -20 °C.

Preparation and characterisation of branched poly (styrene-co-acrylonitrile) via atom transfer radical polymerisation using β-bromoethyl benzene as initiator

Branched poly (styrene-co-acrylonitrile) was prepared by atom transfer radical polymerisation (ATRP) of styrene (St) and acrylonitrile (AN) with the feed composition of 0·5 using β-bromoethyl benzene (BEB) as the initiator and divinylbenzene (DVB) as the branching agent. The polymerisation kinetics and the development of branching were studied by gas chromatography (GC), triple detection size exclusion chromatography (TD-SEC) and proton nuclear magnetic resonance (1H-NMR) spectroscopy. The primary chains with the pendent vinyl groups were formed dominantly at a monomer conversion of less than 50%. The pendent vinyl group participated in the polymerisation, which resulted in the formation of branching structure accompanied by a rapid increase in molecular weight and a broadening in its distribution. The branching chains developed gradually as the molecular weight increased. The copolymers of higher molecular weights had higher branching degree than those of lower molecular weights.

Superhyperfine Interactions of the Nitrogen Donors in 4H SiC Studied by Pulsed ENDOR and TRIPLE ENDOR Spectroscopy

The nitrogen donors residing at quasi-cubic lattice site (Nk) in 4H SiC were investigated by field sweep electron spin echo (FS ESE), pulsed electron nuclear double resonance (ENDOR) and pulsed General TRIPLE ENDOR spectroscopy. The 29Si and 13C superhyperfine lines observed in the FS ESE and ENDOR spectra of Nk in n-type 6H SiC were assigned by pulsed General TRIPLE resonance spectroscopy to the specific carbon (C) and silicon (Si) atoms located in the nearest environment of Nk in 4H SiC. The superhyperfine interaction constants and their relative signs for Nk with 29Si and 13C nuclei located in the nearest-neighbor shells are found from the General TRIPLE ENDOR spectra to be positive for C atoms and negative for Si atoms.

Electronic structure of the nitrogen donors in 6H SiC as studied by pulsed ENDOR and TRIPLE ENDOR spectroscopy

AbstractThe nitrogen (N) donors in 6H SiC were investigated by field sweep electron spin echo (FS ESE), pulsed electron nuclear double resonance (ENDOR) and pulsed General TRIPLE ENDOR spectroscopy. The 29Si and 13C superhyperfine (shf) lines observed in the FS ESE and ENDOR spectra of N in n‐type 6H SiC were assigned by pulsed General TRIPLE resonance spectroscopy to the specific carbon (C) and silicon (Si) atoms located in the environment of N donors residing at two quasi‐cubic lattice sites (Nk1, Nk2) in 6H SiC. As a result, the largest value of the shf interaction was found with Si atoms for the N donors at the hexagonal (Nh) and quasi‐cubic site Nk1, while for Nk2 the largest value of the shf interaction was found with C atoms. It gives us the argument to consider that N substitute different lattice sites in 6H SiC lattice. The relative signs of the shf interaction (shfi) constants for Nk1 and Nk2 with 29Si and 13C nuclei located in the nearest‐neighbor, next nearest‐neighbor, and outer shells are found from the TRIPLE ENDOR spectra to be positive for C atoms and negative for Si atoms. From the comparison of the experimentally obtained shfi constants with the theory, the electronic spin‐density distribution over the 29Si and 13C nuclei located in the nearest neighbor shells of N donors has been obtained taking into account the Kohn–Luttinger interference effect. The position of the conduction band minimum along the ML‐line was determined to be at k0z/kmax = 0.2 ± 0.05.

The np Rydberg series of boron monohydride: l-uncoupling and its evolution for intermediate principal quantum numbers n = 4 to n = 11

Using optical-optical-optical triple-resonance spectroscopy, we assign rotational levels with N = 0-5 in the vibrationless, lower-n, p Rydberg states of (11)BH. We apply the Hill and Van Vleck formulation for energy levels with l = 1 in a Hund's case intermediate between (b) and (d) to gauge the energy separating (1)Π and (1)Σ(+) states with zero rotation for n = 4-11. This energy difference, A(l, ξ), represents the strength of the coupling, ξ, between the electron orbital angular momentum, l, and the internuclear axis, which determines the Λ-splitting constant, q(0). The np series exhibits a large q(0) that increases monotonically with n to reach a magnitude similar to the rotational constant, B(0), by n = 9. For higher principal quantum numbers, Λ ceases to be a good quantum number, and l-uncoupling becomes virtually complete for n > 10.

The Domain 2 of the HCV NS5A Protein Is Intrinsically Unstructured

We present here our current understanding of the NS5A-D2 domain of the hepatitis C virus. Whereas this protein domain is globally unstructured as assessed by macroscopic techniques such as size exclusion chromatography, circular dichroism and homonuclear NMR spectroscopy, high resolution triple resonance spectroscopy allows the identification of a small region of residual structure. This region corresponds moreover to the most conserved sequence over the different genotypes of the virus, underscoring its functional importance. We show that it forms an anchoring point for the host cell cyclophilin prolyl cis/trans isomerase, providing a molecular basis for the use of cyclophilin inhibitors in an antiviral strategy.

Air-stable Curved π-Radical Based on Corannulene: Dynamic Electronic-spin Structure Induced by Temperature-dependent Conformational Changes

A new corannulene-based curved neutral π-radical bearing a tert-butylnitroxide moiety has been synthesized and isolated as an air-stable solid. Direct attachment of the spin centre to the corannulene skeleton gives rise to an extensive spin-delocalization onto the curved π-conjugated system from the nitroxide moiety. This salient electronic feature in the curved neutral radical system and its high stability have allowed us to find a dynamic electronic-spin behaviour induced by the temperature-dependent conformational change of the nitroxide moiety, as studied by liquid-phase variable-temperature electron spin resonance and 1H electron-nuclear double resonance and electron-nuclear-nuclear triple resonance spectroscopies with the help of density functional theory calculations.

NMR assignments of the sylvatic dengue 1 virus envelope protein domain III

Nearly complete backbone and side chain resonance assignments have been obtained for the third domain, residues M289-K400, of the envelope protein from the sylvatic strain (P72-1244) of the dengue 1 virus, containing mutations N336S and E370K, using double- and triple-resonance spectroscopy.

NMR assignments of the yellow fever virus envelope protein domain III

Nearly complete backbone and sidechain resonance assignments have been obtained for the third domain, residues S288-K398, of the envelope protein from the Asibi strain of yellow fever virus using double- and triple-resonance spectroscopy.

Dynamics of dissociative recombination versus electron ejection in single rovibronic resonances of BH

Optical-optical-optical triple resonance spectroscopy isolates transitions to vibrationless Rydberg states of BH with principal quantum numbers from n=7 to 50. Corresponding resonances appear in the excitation spectrum of excited boron atoms produced by the dissociative relaxation of these states. The decay to neutral products occurs on a nanosecond time scale. Yet, corresponding resonances show Fano coupling widths that approach 1 cm-1. Above threshold, spontaneous ionization dominates, but line shapes match for resonances with the same electron orbital quantum numbers built on v+=0 and v+=1 cores. This striking feature-for-feature similarity in predissociation and autoionization line shapes affirms that inelastic electron-cation scattering pathways leading to electron ejection and dissociative recombination proceed through a common continuum.

Structure of FBP11 WW1-PL Ligand Complex Reveals the Mechanism of Proline-rich Ligand Recognition by Group II/III WW Domains

FBP11/HYPA is a mammalian homologue of yeast splicing factor Prp40. The first WW domain of FBP11/HYPA (FBP11 WW1) is essential for preventing severe neurological diseases such as Huntington disease and Rett syndrome and strongly resembles the WW domain of FCA, the essential regulator for flowering time control. We have solved the structure of FBP11 WW1 and a Pro-Pro-Leu-Pro ligand complex, and demonstrated the binding mechanism with mutational analysis using surface plasmon resonance. The overall structure of FBP11 WW1 in the complex form is quite similar to the structures of WW domains from Group I and IV in complexes. In addition, conformation of FBP11 WW1 does not change much upon ligand binding. The binding orientation of the ligand against FBP11 WW1 is the same as that of the Group IV WW domain-ligand complex, but opposite to that of the Group I complex. The ligand interacts with two grooves formed by surface aromatic residues. The Pro and Leu residues in the ligand interact with the grooves and the Loop I region of FBP11 WW1, respectively, which are necessary interactions for binding the ligand. Interestingly, the two aromatic grooves recognize the Pro residues in entirely different manners, which allows FBP11 WW1 to recognize shorter sequences than the SH3 domain. Combined with homology models of other WW domains, the present report shows the detailed mechanism of ligand binding by Group II/III WW domains, and provides information useful in designing drugs to treat neurodegenerative diseases.

Rovibrational characterization of XΣ+2B11H+ by the extrapolation of photoselected high Rydberg series in B11H

Optical-optical-optical triple-resonance spectroscopy of (11)BH isolates high Rydberg states that form series converging to rotational state specific ionization potentials in the vibrational levels of (11)BH(+) from nu(+)=0 through 4. Limits defined by a comprehensive fit of these series to state-detailed thresholds yield rovibrational constants describing the X (2)Sigma(+) state of (11)BH(+). The data provide a first determination of the vibrational-rotational interaction parameter alpha(e)=0.4821 cm(-1) and a more accurate estimate of omega(e)=2526.58 cm(-1) together with the higher-order anharmonic terms omega(e)x(e)=61.98 cm(-1) and omega(e)y(e)=-1.989 cm(-1). The deperturbation and global fit of series to state-detailed limits also yield a precise value of the adiabatic ionization potential of (11)BH of 79 120.3+/-0.1 cm(-1), or 9.810 33+/-1x10(-5) eV. High precision is afforded here by the use of graphical analysis techniques, narrow-bandwidth laser systems, and an analysis of newly observed, high principal quantum number Rydberg states that conform well with Hund's case (d) electron-core coupling limit.

Rotational spectroscopy of the first excited state of the acetylenic C–H stretch of 3-fluoropropyne performed by infrared-Fourier transform microwave–microwave triple-resonance spectroscopy

The rotational spectra of 3-fluoropropyne in the ground and first excited acetylenic C–H stretch vibrational state have been measured. The pure rotational spectrum of the normal species and the 13C isotopomers were measured using FTMW-cwMW double-resonance spectroscopy based on the Autler–Townes (AC Stark) effect. The lineshape properties of this measurement make it possible to determine the transition strength, Δ J-selection rules, and the relative energy ordering of the quantum states. The frequency accuracy of this technique is tested against a previous pure rotational study of 3-fluoropropyne. The rotational spectrum of vibrationally excited state was obtained through IR-FTMW-cwMW methods. In this technique a single-longitudinal-mode pulsed infrared laser source vibrationally excites the acetylenic C–H stretch with J-selectivity. The rotational spectrum of the excited state is then obtained by FTMW and FTMW-cwMW double-resonance methods. The excited-state measurements have a signal-to-noise ratio comparable to the pure rotational spectrum. The residuals in the excited-state fit are larger than those obtained in the ground-state fit. This greater deviation from a standard asymmetric top spectrum is most likely due to weak perturbations to the acetylenic C–H spectrum.

Motional narrowing of the rotational spectrum of trifluoropropyne at 6550 cm(-1) by intramolecular vibrational energy redistribution.

We present the basic principles of dynamic rotational spectroscopy for the highly vibrationally excited symmetric top molecule trifluoropropyne (TFP,CF3CCH). Single molecular eigenstate rotational spectra of TFP were recorded in the region of the first overtone of the nu(1) acetylenic stretching mode at 6550 cm(-1) by infrared-pulsed microwave-Fourier transform microwave triple resonance spectroscopy. The average rotational constant (B) of the highly vibrationally mixed quantum states at 6550 cm(-1) is 2909.33 MHz, a value that is 40 MHz larger than the rotational constant expected for the unperturbed C-H stretch overtone (2869.39 MHz). The average rotational constant and rotational line shape of the molecular eigenstate rotational spectra are compared to the distribution of rotational constants expected for the ensemble of normal-mode vibrational states at 6550 cm(-1) that can interact by intramolecular vibrational energy redistribution (IVR). The normal-mode population distribution at 6550 cm(-1) can be described using a Boltzmann distribution with a microcanonical temperature of 1200 K. At this energy the rotational constant distribution in the normal-mode basis set is peaked at about 2910 MHz with a width of about 230 MHz. The distribution is slightly asymmetric with a tail to the high end. The experimentally measured dynamic rotational spectra are centered at the normal-mode distribution peak; however, the spectral width is significantly narrower (40 MHz) than normal-mode ensemble width (230 MHz). This reduction of the width, along with the Lorentzian shape of the eigenstate rotational spectra when compared to the Gaussian shape of the calculated ensemble distribution, illustrates the narrowing of the spectrum due to IVR exchange. The IVR exchange rate was determined to be 120 ps, about ten times faster than the rate at which energy is redistributed from the v=2 level of the acetylenic stretch.

Coupling of electron orbital motion with rotation in the high Rydberg states of BH.

We have applied optical-optical-optical triple resonance spectroscopy to resolve a system of high Rydberg states in BH that serves quantitatively to characterize a fundamental example of electron-orbital-cation core rotational coupling. The third-color ionization-detected absorption spectrum originating from the photoselected 3s B1Sigma+ Rydberg state with vibrational and total angular momentum quantum numbers, v'=1 and N'=0 consists entirely of vibrationally autoionizing resonances for which final N=1 that converge in series to the BH+v+=1 rotational limits, N+=0, 1, and 2. For series with l=1 converging to N+=0 and 2, Rydberg orbital and rotational angular momenta couple to systematically perturb level energies and distribute lifetime in a well-isolated two-channel rotronic interaction that spans hundreds of wave numbers.