Triple-resonance Techniques Research Articles

Low-lying quasibound rovibrational states of H2 16O**

A complex coordinate scaling (CCS) method is described allowing the quantum chemical computation of quasibound (also called resonance or metastable) rovibrational states of strongly bound triatomic molecules. The molecule chosen to test the method is H2 16O, for which an accurate global potential energy surface, a previous computation of a few resonance states via the complex absorbing potential (CAP) method, and some Feshbach (J = 0, where J is the quantum number characterising overall rotations of the molecule) and shape (J ≠ 0) resonances measured via a state-selective, triple-resonance technique are all available. Characterisation of the computed resonance states is performed via probability density plots based on CCS rovibrational wavefunctions. Such plots provide useful details about the physical nature of the resonance states. Based on the computations and the resonance plots, the following useful facts about the resonance states investigated are obtained: (a) Feshbach resonances are formed by accumulation of a large amount of energy in either the non-dissociative bending or symmetric streching modes, excitations by more than five quanta are not uncommon; (b) there are several resonance states with low and medium bending excitation, the latter are different from the states observed somewhat below dissociation by the same triple-resonance technique; (c) several types of dissociation bahavior can be identified, varying greatly among the states; (d) several pairs of J = 0 and J = 1 Feshbach resonance states can be identified which differ by rigid-rotor type energies; and (e) the lifetimes of the assigned J = 1 rovibrational Feshbach resonances are considerably longer than the lifetimes of their J = 0 vibrational counterparts.

NMR Resonance Assignments of Sparsely Labeled Proteins: Amide Proton Exchange Correlations in Native and Denatured States

Protein NMR assignments of large proteins using traditional triple resonance techniques depends on double or triple labeling of samples with (15)N, (13)C, and (2)H. This is not always practical with proteins that require expression in nonbacterial hosts. Labeling with isotopically labeled versions of single amino acids (sparse labeling) often is possible; however, resonance assignment then requires a new strategy. Here a procedure for the assignment of cross-peaks in (15)N-(1)H correlation spectra of sparsely labeled proteins is presented. It relies on the correlation of proton-deuterium amide exchange rates in native and denatured spectra of the intact protein, followed by correlation of chemical shifts in the spectra of the denatured protein with chemical shifts of sequenced peptides derived from the protein. The procedure is successfully demonstrated on a sample of a protein, Galectin-3, selectively labeled with (15)N at all alanine residues.

Unique Dimeric Structure of BNip3 Transmembrane Domain Suggests Membrane Permeabilization as a Cell Death Trigger

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.

The Rotational Spectrum and Structure of HOOOH.

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The Rotational Spectrum and Structure of HOOOH

Dihydrogen trioxide, HOOOH, which is a species with fundamental importance for understanding the chain formation ability of the oxygen atom, was detected in a supersonic jet by a Fourier transform microwave spectrometer with a pulsed discharge nozzle, together with double resonance and triple resonance techniques. Its precise molecular structure was determined from the experimentally determined rotational constants of HOOOH and its isotopomer, DOOOD. Many of the microwave and millimeter wave transitions can now be accurately predicted, which could be facilitated for remote sensing of the molecule to elucidate its roles in various chemical processes.

Backbone 1H, 15N, and 13C Resonance Assignment of HP1242 from Helicobacter pylori

One of the small proteins from Helicobacter pylori, HP1242, was investigated by the solution nuclear magnetic resonance (NMR) spectroscopy. HP1242 is known as a 76-residue conserved hypothetical protein and its function cannot be identified based on sequence homology. Here, the results of the backbone (1)H, (15)N, and (13)C resonance assignments of the HP1242 are reported using double- and triple-resonance techniques. About 95 % of all of the (1)HN, (15)N, (13)CO, (13)Calpha, and (13)Cbeta resonances that cover 75 non-Proline residues of the 76 residues are clarified through sequential- and specific- assignments. In addition, three helical regions were clearly identified on the basis of the resonance assignments.

Ubiquitin Manipulation by an E2 Conjugating Enzyme Using a Novel Covalent Intermediate

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.

Backbone 1H, 15N, and 13C Resonance Assignments of the Helicobacter pylori Acyl Carrier Protein

One of the small proteins from Helicobacter pylori, acyl carrier protein (ACP), was investigated by NMR. ACP is related to various cellular processes, especially with the biosynthesis of fatty acid. The basic NMR resonance assignment is a prerequisite for the validation of a heterologous protein interaction with ACP in H. pylori. Here, the results of the backbone (1)H, (15)N, and (13)C resonance assignments of the H. pylori ACP are reported using double- and triple-resonance techniques. About 97% of all of the (1)HN, (15)N, (13)CO, (13)Calpha, and (13)Cbeta resonances that cover 76 of the 78 non-proline residues are clarified through sequential- and specific- assignments. In addition, four helical regions were clearly identified on the basis of the resonance assignments.

Characterization of Isomeric Structures in a Mixture of Organosilanes Using Multidimensional NMR

Triple resonance 1H/13C/29Si three-dimensional nuclear magnetic resonance methods were used to characterize the isomeric structures in a mixture of isomeric disilylated 4-vinyl-1-cyclohexene. Triple resonance techniques when used together with pulsed field gradient coherence selection permit selective detection of the structure fragments of 1H−13C−29Si spin systems. Three-frequency correlation provides a method of separating the resonances from a complex mixture of isomers without physical separation of the components. Many signals that are not resolvable in 1D NMR or 2D NMR spectra can be seen with the aid of triple resonance 3D NMR techniques. The experiment was done in three ways to selectively detect correlations between 29Si resonances and CHn groups which are one, two, and three bonds away. The combined data from the three experiments provide unambiguous atomic connectivity information.

Autler–Townes splitting and the AC Stark effect in nonpolar molecules: Prospects for all-optical alignment

This paper describes an extension of the familiar coherence effects from atomic systems to the molecular regime. Such effects are inherent in the interaction of multiple laser fields with molecular systems. We have observed Autler–Townes splitting and the AC Stark shift in diatomic Lithium using the continuous wave all-optical triple resonance (AOTR) techniques. By using the Autler–Townes effect, we have partially resolved the magnetic sublevels of a molecular rovibrational level in a Doppler broadened sample, allowing all-optical alignment of the angular momentum in excited states of nonpolar molecules. The Autler–Townes effect in a molecular system extends the rovibrational state selectivity of the AOTR excitation technique to magnetic sublevels. PACS Nos.: 33.40tf, 42.50Hz

Characterization of Sn-Containing Polymer Chain Ends of Polybutadiene Using 1H/13C/119Sn Triple-Resonance 3D-NMR

1H/13C/119Sn triple-resonance 3D-NMR spectroscopy is used to characterize the tin-containing structures in the organotin polymer n-Bu3SnPBD (PBD = polybutadiene). Triple-resonance techniques, when used together with pulsed field gradient coherence selection, permit selective detection of the structure fragments near 119Sn atom while removing intense signals of the PBD backbone from the spectrum. Three frequency dimensions provide enormous spectral dispersion, permitting the resolution of many signals that are not observable in 1D-NMR spectra. The simultaneous correlation of resonances from three nuclei provides a large amount of unambiguous atomic connectivity information. The capabilities of modern instruments are such that it is possible to perform these experiments without the benefit of isotopic labeling, despite the low concentration of chain ends and the low occurrence of fragments with 1H, 13C, and 119Sn isotopes.

Mapping the effects of metal ion reduction and substrate analog binding to Fe-superoxide dismutase by NMR spectroscopy

Fe-containing superoxide dismutase (FeSOD) is an excellent model for studies of the role of the protein in shaping redox catalytic activity in metalloenzymes. In order to use NMR spectroscopy to monitor the protons in FeSOD, we have assigned all the observable backbone resonances in the HN heteronuclear single quantum correlation spectrum (HN-HSQC), in both of the reduced and oxidized states. This task required 2H, 13C and 15N triple labeling and quadruple resonance techniques due to FeSOD's molecular weight of 42 kDa and paramagnetic high-spin active site Fe. The unobserved and unassigned residues of FeSOD are accounted for by paramagnetic relaxation and slow back-exchange of solvent protons into backbone HN positions. Of FeSOD's 192 residues, we have previously assigned resonances for 118 in the oxidized state and now 141 in the reduced state. For residues > 14 Å from Fe, resonances are observable in both oxidation states. Relatively small chemical shift changes were found to accompany Fe2+ oxidation, but these extend throughout the protein. Specific binding of the substrate analog F− to Fe2+SOD was found to involve residues near Tyr34, consistent with participation of this residue in substrate binding. This represents the first location of substrate binding to Fe2+SOD. Additional residues at the dimer interface are sensitive to a variety of anions in both of FeSOD's oxidation states and also Fe oxidation, consistent with either interaction with substrate or domain movement associated with substrate binding and metal ion oxidation. Copyright © 2000 John Wiley & Sons, Ltd.

NMR structure determination of the tetramerization domain of the Mnt repressor: An asymmetric alpha-helical assembly in slow exchange.

The structure and dynamics of the chymotryptic tetramerization domain of the Mnt repressor of Salmonella bacteriophage P22 have been studied by NMR spectroscopy. Two sets of resonances (A and B) were found, representing the asymmetry within the homotetramer. Triple-resonance techniques were used to obtain unambiguous assignments of the A and B resonances. Intra-monomeric NOEs, which were distinguished from the inter-monomeric NOEs by exploiting (13)C/(15)N-filtered NOE experiments, demonstrated a continuous alpha-helix of approximately seven turns for both the A and B monomers. The asymmetry facilitated the interpretation of inter-subunit NOEs, whereas the antiparallel alignment of the subunits allowed further discrimination of inter-monomeric NOEs. The three-dimensional structure revealed an unusual asymmetric packing of a dimer of two antiparallel right-handed intertwined coiled alpha-helices. The A and B forms exchange on a timescale of seconds by a mechanism that probably involves a relative sliding of the two coiled coils. The amide proton solvent exchange rates demonstrate a stable tetrameric structure. The essential role of Tyr 78 in oligomerization of Mnt, found by previous mutagenesis studies, can be explained by the many hydrophobic and hydrogen bonding interactions that this residue participates in with adjacent monomers.

MEASUREMENT OF THE HYPERFINE STRUCTURE OF ANTIPROTONIC HELIUM

This paper discusses the level splitting of the antiprotonic helium atomcule, an exotic atom consisting of a helium nucleus, an electron, and an antiproton which occupies highly excited states with angular momentum of L ≈ 30–35. The observation of a splitting in a laser transition between two levels is presented, and an experiment in preparation at the forthcoming AD facility at CERN is described which aims at directly measuring the level splitting by using a 2-laser microwave triple resonance technique. This experiment has the potential to determine the magnetic moment of the antiproton with higher precision than currently known.

3D Accordion Spectroscopy for Measuring15N and13CO Relaxation Rates in Poorly Resolved NMR Spectra

An experimental approach for the measurement of nuclear magnetic spin relaxation rate constants that combines triple-resonance techniques and accordion spectroscopy is described. Pulse sequences are discussed for the measurement of backbone15N and13COR1relaxation rate constants. The three-dimensional HNCO triple-resonance technique is employed to gain improved spectral resolution over conventional two-dimensional methods by frequency labeling both the15N and13CO spins. Accordion spectroscopy is used to reduce the dimensionality of the relaxation experiment. The “negative-time accordion” approach (A. M. Mandel and A. G. Palmer (1994),J. Magn. Reson. A110, 62–72) is used for extracting rate constants from thet1interferograms. The experiments are demonstrated using a13C/15N isotopically enriched sample of the third fibronectin type III domain of human tenascin.

Accurate measurements of electric quadrupole hyperfine interactions of very dilute spins in magnetic solids

The application of double and triple resonance techniques to enhance signals in quadrupole interaction — nuclear magnetic resonance on oriented nuclei spectroscopy, is illustrated for the antiferromagnet ( 54Mn)MnBr 2-4H 2O. Unusual shifts of the quadrupolar split, higher order, ν ±2, ±1 subresonance, comparable to the NMRON linewidth, are observed and explained.

NMR studies of Borrelia burgdorferi OspA, a 28 kDa protein containing a single-layer beta-sheet.

The crystal structure of outer surface protein A (OspA) from Borrelia burgdorferi contains a single-layer beta-sheet connecting the N- and C-terminal globular domains. The central beta-sheet consists largely of polar amino acids and it is solvent-exposed on both faces, which so far appears to be unique among known protein structures. We have accomplished nearly complete backbone H, C and N and C beta/H beta assignments of OspA (28 kDa) using standard triple resonance techniques without perdeuteration. This was made possible by recording spectra at a high temperature (45 degrees C). The chemical shift index and 15N T1/T2 ratios show that both the secondary structure and the global conformation of OspA in solution are similar to the crystal structure, suggesting that the unique central beta-sheet is fairly rigid.

Reduction in the amide hydrogen exchange rates of an anti-lysozyme Fv fragment due to formation of the Fv-Lysozyme complex

The Fv fragment of the monoclonal antibody D1.3 was expressed in bacteria. Standard triple resonance techniques were used to obtain the NMR resonance assignments for 211 out of 215 backbone 15N/NH atoms for D1.3 Fv. Using these assignments, hydrogen exchange rates are measured for 82 amide hydrogen atoms in D1.3 Fv free and bound to hen egg-white lysozyme. Upon binding to antigen, exchange rates are decreased for residues throughout the Fv. Many of these residues are located remote from the site of interaction with the antigen. These changes are larger than previously observed for the antigen portion of the complex. Evidently, the β-sheet structure of the Fv propagates the effects of binding more efficiently than the antigen. These effects are compared between the three different polypeptide chains that make up the complex. These data suggest that reduced dynamics are a general feature of antibody binding to antigen.

Mimicking primary processes in photosynthesis—covalently linked porphyrin quinones

Porphyrin quinones (P-Qs), covalently linked via different aliphatic bridges, have been synthesized and studies in their (porphyrin) cationic and (semiquinone) anionic radical states by EPR, ENDOR and TRIPLE resonance techniques. Electron transfer (ET) from the porphyrin donor to the quinone acceptor could be observed by time-resolved picosecond fluorescence spectroscopy (singlet ET) and by time-resolved EPR spectroscopy (triplet ET) in isotropic fluid solution and in anisotropic media (liquid crystals and reversed micelles). Steady-state in situ photoexcitation of P-Qs in CTAB cationic reversed micelles yielded the corresponding semiquinone radical anions. In TRITON X-100 reversed micelles both the radical cation of the porphyrin and the radical anion of the semiquinone could be detected, which occured in complete emission. In covalently linked porphyrin flavins ET from the photoexcited porphyrin fragment to the flavin and, in addition, energy transfer from the photoexcited flavin to the porphyrin could be observed.

Assignment of the backbone carbonyl resonances in 15N-labelled proteins with 13C at natural abundance by a 2D triple-resonance correlation technique

A 2D NMR experiment for assignment of backbone carbon resonances in small and medium-sized (15)N-labelled proteins with (13)C at natural abundance is presented. The experiment is a two-dimensional variant of the HNCO triple-resonance experiment and is demonstrated by application to a 6 kDa protein at relatively low concentration (2 mM) and temperature (30°C). The experiment is particularly suitable for assignment of carbonyl resonances.