The two-dimensional IR nonlinear spectroscopy of a cyclic penta-peptide in relation to its three-dimensional structure

A form of two-dimensional (2D) vibrational spectroscopy, which uses two ultrafast IR laser pulses, is used to examine the structure of a cyclic penta-peptide in solution. Spectrally resolved cross peaks occur in the off-diagonal region of the 2D IR spectrum of the amide I region, analogous to those in 2D NMR spectroscopy. These cross peaks measure the coupling between the different amide groups in the structure. Their intensities and polarizations relate directly to the three-dimensional structure of the peptide. With the help of a model coupling Hamiltonian, supplemented by density functional calculations, the spectra of this penta-peptide can be regenerated from the known solution phase structure. This 2D-IR measurement, with an intrinsic time resolution of less than 1 ps, could be used in all time regimes of interest in biology.

Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by 13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680.+ primary donor chlorophyll

We report 13C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo- CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance 13C provide information on the electronic structure of the primary electron donor P680 (chlorophyll a molecules absorbing around 680 nm) and on the pz spin density pattern in its oxidized form, P680⨥. Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P680⨥. PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P680 by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.

Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond

Single-stranded regions in RNA secondary structure are important for RNA–RNA and RNA–protein interactions. We present a probability profile approach for the prediction of these regions based on a statistical algorithm for sampling RNA secondary structures. For the prediction of phylogenetically-determined single-stranded regions in secondary structures of representative RNA sequences, the probability profile offers substantial improvement over the minimum free energy structure. In designing antisense oligonucleotides, a practical problem is how to select a secondary structure for the target mRNA from the optimal structure(s) and many suboptimal structures with similar free energies. By summarizing the information from a statistical sample of probable secondary structures in a single plot, the probability profile not only presents a solution to this dilemma, but also reveals ‘well-determined’ single-stranded regions through the assignment of probabilities as measures of confidence in predictions. In antisense application to the rabbit β-globin mRNA, a significant correlation between hybridization potential predicted by the probability profile and the degree of inhibition of in vitro translation suggests that the probability profile approach is valuable for the identification of effective antisense target sites. Coupling computational design with DNA–RNA array technique provides a rational, efficient framework for antisense oligonucleotide screening. This framework has the potential for high-throughput applications to functional genomics and drug target validation.

Induced fit DNA recognition by a minor groove binding analogue of Hoechst 33258: fluctuations in DNA A tract structure investigated by NMR and molecular dynamics simulations

NMR analysis and molecular dynamics simulations of d(GGTAATTACC)2 and its complex with a tetrahydropyrimidinium analogue of Hoechst 33258 suggest that DNA minor groove recognition in solution involves a combination of conformational selection and induced fit, rather than binding to a preorganised site. Analysis of structural fluctuations in the bound and unbound states suggests that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the minor groove resulting in groove narrowing through: (i) changes in base step parameters, including increased helical twist and propeller twist; (ii) changes to the sugar–phosphate backbone conformation to engulf the bound ligand; (iii) suppression of bending modes at the TpA steps. In contrast, the geometrical arrangement of hydrogen bond acceptors on the groove floor appears to be relatively insensitive to DNA conformation (helical twist and propeller twist). We suggest that effective recognition of DNA sequences (in this case an A tract structure) appears to depend to a significant extent on the sequence being flexible enough to be able to adopt the geometrically optimal conformation compatible with the various binding interactions, rather than involving ‘lock and key’ recognition.

Multiple four-stranded conformations of human telomere sequence d(CCCTAA) in solution

By detailed NMR analysis of a human telomere repeating unit, d(CCCTAA), we have found that three distinct tetramers, each of which consists of four symmetric single-strands, slowly exchange in a slightly acidic solution. Our new finding is a novel i-motif topology (T-form) where T4 is intercalated between C1 and C2 of the other duplex. The other two tetramers have a topology where C1 is intercalated between C2 and C3 of the other parallel duplex, resulting in the non-stacking T4 residues (R-form), and a topology where C1 is stacked between C3 and T4 of the other duplex (S-form). From the NMR denaturation profile, the R-form is the most stable of the three structures in the temperature range of 15–50°C, the S-form the second and the T-form the least stable. The thermodynamic parameters indicate that the T-form is the most enthalpically driven and entropically opposed, and its population is increased with decreasing temperature. The T-form structure determined by restrained molecular dynamics calculation suggests that inter-strand van der Waals contacts in the narrow grooves should contribute to the enthalpic stabilization of the T-form.

Solution 19F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

19F nuclear Overhauser effects (NOEs) between fluorine labels on the cytoplasmic domain of rhodopsin solubilized in detergent micelles are reported. Previously, high-resolution solution 19F NMR spectra of fluorine-labeled rhodopsin in detergent micelles were described, demonstrating the applicability of this technique to studies of tertiary structure in the cytoplasmic domain. To quantitate tertiary contacts we have applied a transient one-dimensional difference NOE solution 19F NMR experiment to this system, permitting assessment of proximities between fluorine labels specifically incorporated into different regions of the cytoplasmic face. Three dicysteine substitution mutants (Cys-140–Cys-316, Cys-65–Cys-316, and Cys-139–Cys-251) were labeled by attachment of the trifluoroethylthio group through a disulfide linkage. Each mutant rhodopsin was prepared (8–10 mg) in dodecylmaltoside and analyzed at 20°C by solution 19F NMR. Distinct chemical shifts were observed for all of the rhodopsin 19F labels in the dark. An up-field shift of the Cys-316 resonance in the Cys-65–Cys-316 mutant suggests a close proximity between the two residues. When analyzed for 19F-19F NOEs, a moderate negative enhancement was observed for the Cys-65–Cys-316 pair and a strong negative enhancement was observed for the Cys-139–Cys-251 pair, indicating proximity between these sites. No NOE enhancement was observed for the Cys-140–Cys-316 pair. These NOE effects demonstrate a solution 19F NMR method for analysis of tertiary contacts in high molecular weight proteins, including membrane proteins.

The structure of the human βII-tryptase tetramer: Fo(u)r better or worse

Tryptases, the predominant serine proteinases of human mast cells, have recently been implicated as mediators in the pathogenesis of allergic and inflammatory conditions, most notably asthma. Their distinguishing features, their activity as a heparin-stabilized tetramer and resistance to most proteinaceous inhibitors, are perfectly explained by the 3-Å crystal structure of human βII-tryptase in complex with 4-amidinophenylpyruvic acid. The tetramer consists of four quasiequivalent monomers arranged in a flat frame-like structure. The active centers are directed toward a central pore whose narrow openings of approximately 40 Å × 15 Å govern the interaction with macromolecular substrates and inhibitors. The tryptase monomer exhibits the overall fold of trypsin-like serine proteinases but differs considerably in the conformation of six surface loops arranged around the active site. These loops border and shape the active site cleft to a large extent and form all contacts with neighboring monomers via two distinct interfaces. The smaller of these interfaces, which is exclusively hydrophobic, can be stabilized by the binding of heparin chains to elongated patches of positively charged residues on adjacent monomers or, alternatively, by high salt concentrations in vitro. On tetramer dissociation, the monomers are likely to undergo transformation into a zymogen-like conformation that is favored and stabilized by intramonomer interactions. The structure thus provides an improved understanding of the unique properties of the biologically active tryptase tetramer in solution and will be an incentive for the rational design of mono- and multifunctional tryptase inhibitors.

New methods of structure refinement for macromolecular structure determination by NMR

Recent advances in multidimensional NMR methodology have permitted solution structures of proteins in excess of 250 residues to be solved. In this paper, we discuss several methods of structure refinement that promise to increase the accuracy of macromolecular structures determined by NMR. These methods include the use of a conformational database potential and direct refinement against three-bond coupling constants, secondary 13C shifts, 1H shifts, T1/T2 ratios, and residual dipolar couplings. The latter two measurements provide long range restraints that are not accessible by other solution NMR parameters.

Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis. Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry. In contrast, analysis of [Met(O)45]4OT and (desPro1)4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT.

Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation.

The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles.

Direct structure analysis in protein electron crystallography: crystallographic phases for halorhodopsin to 6-A resolution.

The crystal structure of halorhodopsin was determined in (centrosymmetric) projection to 6-A resolution by direct methods that use only the amplitudes of the electron diffraction pattern. A multisolution technique was used to generate initial 15-A-resolution basis sets, and after selection of the best phase set (by the closest match of magnitude of Eobs and magnitude of Ecalc), annealing of individual reflections was used to improve its accuracy. The Sayre equation was then used to expand the phase terms to 10 A, followed again by phase annealing. A final expansion with the Sayre equation enlarged this corrected phase set to 6 A. When the condition of density flatness was used to locate the best phase solution after each extension, a final structure could be observed that was quite similar to the one found earlier by analysis of electron micrographs.

Determination of the angle between the anticodon and aminoacyl acceptor stems of yeast phenylalanyl tRNA in solution.

A principal feature of the crystal structures of tRNAs is an L-shaped tertiary conformation in which the aminoacyl acceptor stem and the anticodon stem are approximately perpendicular. However, the anticodon-acceptor interstem angle has not been precisely quantified in solution for any tRNA. Such a determination would represent an important test of the predicted global conformation of tRNAs in solution. To this end, we have constructed a yeast tRNA(Phe) heteroduplex RNA molecule in which the anticodon and acceptor stems of the tRNA have each been extended by approximately 70 base pairs. A comparison of the rotational decay times of the heteroduplex molecule and a linear control yields an interstem angle of 89 +/- 4 degrees in 4 mM magnesium chloride/100 microM spermine hydrochloride, essentially identical to the corresponding angle observed in the crystal under similar buffer and temperature conditions. The current approach is applicable to the study of a wide variety of RNA molecules that possess elements of nonhelical structure.

Applications of EPR with an Emphasis on Tau Fibril Structure

Substances containing unpaired electrons have been studied by electron paramagnetic resonance (EPR) for nearly 70 years. With continual development and enhancement of EPR techniques, questions have arisen regarding optimum method selection for a given sample based on its properties. In this work, radiation defects, natural lattice defects, solid organic radicals, radicals in solution, and spin-labeled proteins were analyzed using CW, pulse, and rapid scan EPR to compare methods. Studies of solid BDPA, EOe in quartz, Ns0 in diamond, and a-Si:H, showed that rapid scan could overcome many obstacles presented by other techniques, cementing rapid scan as an effective alternative to CW and pulse methods. Relaxation times of six nitroxide radicals were characterized from 0.25-34 GHz, guiding synthesis of improved nitroxides for in vivo imaging experiments. Processes contributing to T1 of DPPH in polystyrene were found through variable temperature measurements at X- and Q-band, resolving previously-reported discrepancies in relaxation properties and providing new insight into this commonly-used standard. In the history of EPR, the study of proteins is relatively new. Double electron-electron resonance (DEER) has emerged as a powerful technique for the study of amyloid fibrils, a class of protein aggregates implicated in a number of neurodegenerative disorders. Microtubule-associated protein tau forms fibrils linked to Alzheimerfs disease through seeded conversion of monomer. Self-assembly is mediated by the microtubule binding repeats in tau, and there are either three or four repeats present depending on the isoform. DEER was used to show that filaments of 3R and 4R tau are conformationally distinct and that 4R fibrils adopt a heterogeneous mixture of conformations. Populations of 4R fibril conformations, which were independently validated using a model system, can be modulated by introduction of mutations to the primary sequence or by varying fibril growth conditions. These findings provided unprecedented insights into the seed selection of tau monomers and established conformational compatibility as an important driving force in tau fibril propagation. Lastly, DEER acquisition was improved through addition of paramagnetic metal to spin-labeled protein, decreasing collection time, and through use of a novel spin label with increased T2, thereby lengthening the available acquisition window.

Spin alignment of extra electrons in K-phenanthrene clusters taken from the crystalline tripotassium-intercalated phenanthrene structure

The appearance of ferromagnetic correlations among π electrons of phenanthrene (C14H10) molecules in the herringbone structure is proven for K doped clusters both by ab initio quantum-chemistry calculations and by the direct solution of the many-body Pariser-Parr-Pople Hamiltonian. Magnetic ground states are predicted for one or three additional electrons per phenanthrene molecule. These results are a consequence of the small overlap between the lowest unoccupied molecular orbitals (and lowest unoccupied molecular orbitals + 1) of neutral neighboring phenanthrene molecules, which makes the gain in energy by delocalization similar to the corresponding increase due to the Coulomb interaction.

Synthesis of Robust Hierarchical Silica Monoliths by Surface-Mediated Solution/Precipitation Reactions over Different Scales: Designing Capillary Microreactors for Environmental Applications

A synthetic procedure to prepare novel materials (surface-mediated fillings) based on robust hierarchical monoliths is reported. The methodology includes the deposition of a (micro- or mesoporous) silica thin film on the support followed by growth of a porous monolithic SiO2 structure. It has been demonstrated that this synthesis is viable for supports of different chemical nature with different inner diameters without shrinkage of the silica filling. The formation mechanism of the surface-mediated fillings is based on a solution/precipitation process and the anchoring of the silica filling to the deposited thin film. The interaction between the two SiO2 structures (monolith and thin film) depends on the porosity of the thin film and yields composite materials with different mechanical stability. By this procedure, capillary microreactors have been prepared and have been proved to be highly active and selective in the total and preferential oxidation of carbon monoxide (TOxCO and PrOxCO).