Nuclear Overhauser Enhancement Spectroscopy Spectra Research Articles

An improved, time-efficient approach to extract accurate distance restraints for NMR2 structure calculation.

Exact nuclear Overhauser enhancement (eNOE) yields highly accurate, ensemble averaged H-H distance restraints with an accuracy of up to 0.1 Å for the multi-state structure determination of proteins as well as for nuclear magnetic resonance molecular replacement (MR) to determine the structure of the protein-ligand interaction site in a time-efficient manner. However, in the latter application, the acquired eNOEs lack the obtainable precision of 0.1 Å because of the asymmetrical nature of the filtered nuclear Overhauser enhancement spectroscopy (NOESY) experiment used in MR. This error is further propagated to the eNOE equations used to fit and extract the distance restraints. In this work, a new analysis method is proposed to obtain inter-molecular distance restraints from the filtered NOESY spectrum more accurately and intuitively by dividing the NOE cross peak by the corresponding diagonal peak of the ligand. The method termed diagonal-normalised eNOEs was tested on the data acquired by on the complex of PIN1 and a small, weak-binding phenylimidazole fragment. MR calculations performed using the distances derived from diagonal-normalised eNOEs yielded the right orientation of the fragment in the binding pocket and produced a structure that more closely resembles the benchmark X-ray structure (2XP6) with an average heavy-atom root-mean-square deviation (RMSD) of 1.681 Å with respect to it, when compared to the one produced with traditional MR with an average heavy atom RMSD of 3.628 Å. This is attributed to the higher precision of the evaluated distance restraints.

Interaction of Phenyl-Substituted Diazaperylenium with Cucurbit[n]uril: Complex versus Supramolecular Polymer.

A phenyl-substituted diazaperylenediium dication (DAP) was synthesized that shows concentration-dependent aggregation in water. The host–guest complexation of DAP was studied with the macrocyclic host cucurbit[n]uril (n = 7, 8) in water. With CB[7], it forms a 1:2 complex by placing two aryl substituents inside the CB[7]; whereas, with CB[8], a supramolecular polymer is formed. The resulting complex and supramolecular polymer have been characterized by ITC (isothermal titration calorimetry), ESI-MS (electrospray ionization mass spectrometry), 1H NMR (proton nuclear magnetic resonance), 2D NOESY (two-dimensional nuclear Overhauser enhancement spectroscopy), and DOSY (diffusion-ordered spectroscopy) spectra.

Fully automated assignment of methyl resonances of a 36 kDa protein dimer from sparse NOESY data

High-resolution solution-state NMR spectroscopy studies of large proteins typically require uniform deuteration of the system and selective protonation and isotope labelling of methyl groups. Under such circumstances, the assignment of methyl resonances presents a considerable experimental challenge and automation of the process using computational algorithms has been actively sought. Through-space connectivities between the labelled methyl groups can be established through nuclear Overhauser enhancement spectroscopy (NOESY). If a high-resolution structure of the system is available, the sparse connectivity restraints derived from this information enable structure-based methyl resonance assignment. Here, we outline a protocol for full automation of the methyl resonance assignment process using the CYANA software package. We tested the protocol on three-dimensional (3D) 13C/13C-separated NOESY spectra of a dimer of regulatory chains of aspartate transcarbamoylase (ATCase-r2). We used CYPICK to detect NOE signals, followed by automatic resonance assignment with FLYA. On this dataset, FLYA generated highly similar results using either automatically or manually generated peak lists, confidently assigning ∼60% of the methyl groups with high accuracy (95 ± 2% correctness). We compared this performance to two alternative automatic methyl assignment protocols, MAP-XSII and FLAMEnGO2.0, both of which, similarly to FLYA, support unassigned NOESY peak lists as input.

Further Study on Chemical Constituents of Parnassia wightiana Wall: Four New Dihydro-β-agarofuran Sesquiterpene Polyesters.

Four new (1–4), along with six known (5–10) dihydro-β-agarofuran sesquiterpene polyesters were isolated from the whole plants of Parnassia wightiana. The new compounds were structurally elucidated through spectroscopic analysis including UV (Ultraviolet Spectrum), IR (Infrared Spectrum), 1H-NMR (1Hydrogen-Nuclear Magnetic Resonance), 13C-NMR (13Carbon-Nuclear Magnetic Resonance), DEPT (Distortionless Enhancement by Polarization Transfer), 1H-1H COSY (1H-1H Correlation Spectroscopy), HSQC (Heteronuclear Single Quantum Coherence), HMBC (Heteronuclear Multiple Bond Correlation), NOESY (Nuclear Overhauser Enhancement Spectroscopy) and HR-MS (High Resolution Mass Specttrum) and their absolute configurations were proposed by comparison of NOESY spectra and specific optical rotations with those of known compounds and biosynthesis grounds. Compound 2 is the first sesquiterpene alkaloid isolated from this plant. New compounds 1–4 exhibited some cytotoxic activities against NB4, MKN-45 and MCF-7 cells at 20 μM and of which 4 showed the highest activity against NB4 and MKN-45 cells with inhibition rates of 85.6% and 30.5%, respectively.

NMR characterization of 1,1′‐binaphthyl‐2,2′‐diyl hydrogen phosphate binding to chiral molecular micelles

NMR spectroscopy was used to characterize the binding of the chiral compound 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (BNP) to five molecular micelles with chiral dipeptide headgroups. Molecular micelles have covalent linkages between the surfactant monomers and are used as chiral mobile phase modifiers in electrokinetic chromatography. Nuclear overhauser enhancement spectroscopy (NOESY) analyses of (S)-BNP:molecular micelle mixtures showed that in each solution the (S)-BNP interacted predominately with the N-terminal amino acid of the molecular micelle's dipeptide headgroup. NOESY spectra were also used to generate group binding maps for (S)-BNP:molecular micelle mixtures. In these maps, percentages are assigned to the (S)-BNP protons to represent the relative strengths of their interactions with a specified molecular micelle proton. All maps showed that (S)-BNP inserted into a previously reported chiral groove formed between the molecular micelle's dipeptide headgroup and hydrocarbon chain. In the resulting intermolecular complexes, the (S)-BNP protons nearest to the analyte phosphate group were found to point toward the N-terminal Halpha proton of the molecular micelle headgroup. Finally, pulsed field gradient NMR diffusion experiments were used to measure association constants for (R) and (S)-BNP binding to each molecular micelle. These K values were then used to calculate the differences in the enantiomers' free energies of binding, Delta(DeltaG). The NMR-derived Delta(DeltaG) values were found to scale linearly with electrokinetic chromatography (EKC) chiral selectivities from the literature.

Probing the Interactions of Early Polyketide Intermediates with the Actinorhodin ACP from S. coelicolor A3(2)

Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. Seven thiol ester and thiol ether derivatives of the actinorhodin (act) PKS ACP from Streptomyces coelicolor have been prepared and structurally characterised by NMR to gain insight into ACP-intermediate interactions. Holo ACP synthase has been used to prepare early-stage ACP intermediates of polyketide biosynthesis (holo ACP, acetyl ACP, and malonyl ACP) from the respective coenzyme A derivatives. A synthetic route to stabilised thiol ether ACPs was developed and applied to the preparation of stable 3-oxobutyl and 3,5-dioxohexyl ACP as diketide and triketide analogues. No interaction between the protein and the acyl phosphopantetheine moieties of acetyl, malonyl, or 3-oxobutyl ACP was detected. Analysis of (1)H-(15)N heteronuclear single quantum coherence and nuclear Overhauser enhancement spectroscopy spectra for the triketide ACP revealed exchange between a major ('Tri', 85%) and a minor protein conformer in which the polyketide interacts with the protein ('Tri(*)', 15%). Act ACP was also derivatised with butyryl, hexanoyl, and octanoyl groups. The corresponding NMR spectra showed large chemical shift perturbations centred on helices II and III, indicative of acyl chain binding and significant structural rearrangement. Unexpectedly, butyryl act ACP showed almost identical backbone (1)H-(15)N chemical shifts to Tri(*), suggesting comparable structural changes that might provide insight into the structurally uncharacterised polyketide bound form. Furthermore, butyryl ACP itself underwent slow conformational exchange with a second minor conformer (But(*)) with almost identical backbone chemical shifts to octanoyl act ACP. High-resolution NMR structures of these acylated forms revealed that act ACP was able to undergo dramatic conformational changes that exceed those seen in FAS ACPs. When compared to E. coli FAS ACP, the substrate binding pocket of the act PKS ACP has three specific amino acid substitutions (Thr39/Leu45, Ala68/Leu74, and Leu42/Thr48) that alter the size, shape, and location of this cavity. These conformational changes may play a role in protein-protein recognition and assist the binding of bulky polyketide intermediates.

Aggregation behavior of sodium deoxycholate and its interaction with cetyltrimethylammonium bromide in aqueous solution studied by NMR spectroscopy

In pure sodium deoxycholate (NaDC) dilute solution, the transverse relaxation times (T (2)) of nearly all proton signals of NaDC obey single-exponential decay, with exception for proton at 3-position (H3) that decays in a two-exponential manner. The two components with different mobility of H3 of NaDC indicate that the molecules are probably associated in head-to-tail pairs via hydrogen bonds in dilute solution. In the mixed NaDC/cetyltrimethylammonium bromide (CTAB) solutions, the T (2) values of interested NaDC protons are far less than those in pure NaDC solution, especially when the concentrations of the two components are close to equal-molar. The results of self-diffusion coefficients and the chemical shifts confirm further that the strong interaction occurs between the two components in mixed solutions, especially for equal-molar condition. The arrangement of the mixed aggregates can be speculated for the cross-peaks of proton pairs occurring between NaDC and CTAB molecules in two-dimensional and rotating frame nuclear Overhauser enhancement spectroscopy spectra.

Chemometric Approach in Quantification of Structural Identity/Similarity of Proteins in Biopharmaceuticals

We present a chemometrics study in which we show the identity or degree of similarity of 3D protein structures of various G-CSF (Granulocyte Colony-Stimulating Factor) isolates. The G-CSF isolates share the same amino acid sequence, but the preparation was carried out by somehow diverse technologies. The comparison of 3D structures was made on the basis of 2D NMR NOESY (Nuclear Overhauser Enhancement Spectroscopy) spectra of proteins. In searching for the most appropriate criteria to determine the identity or degree of similarity of selected spectral regions of different isolates, two methods for quantitative evaluation of identity/similarity were used. The first method compares all peaks in the two investigated protein spectral regions; the extent of peaks that overlap is determined. The second method includes spectral invariants originating from graph theory. The criteria of identity/similarity were calculated from graphs, derived from a collection of up to 200 peaks of investigated 2D NMR spectral region. The peaks were linked into a graph according to the sequential nearest neighborhoods. According to the first method all peaks were relevant, considering that spectral noise was previously removed; the largest similarity was found between the protein of a commercially available G-CSF drug and one of the three new isolates produced in the laboratory. The second method indicated that the pairwise similarity of the three new isolates is larger than the similarity of any of the new isolates with the commercially available drug. This is an expected result taking into account that the new isolates are produced by the same technology, while the commercial product has additives for long-term storage that could not be completely compensated. The proposed measure of similarity may help the developers of biosimilar products to optimize the controllable parameters of the production technology and eventually to argue the identity of the new isolate in comparison with the originator commercial product.

Hydration of POPC bilayers studied by 1H-PFG-MAS-NOESY and neutron diffraction.

The stability of lipid bilayers is ultimately linked to the hydrophobic effect and the properties of water of hydration. Magic angle spinning (MAS) nuclear Overhauser enhancement spectroscopy (NOESY) with application of pulsed magnetic field gradients (PFG) was used to study the interaction of water with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers in the fluid phase. NOESY cross-relaxation between water and polar groups of lipids, but also with methylene resonances of hydrophobic hydrocarbon chains, has been observed previously. This observation led to speculations that substantial amounts of water may reside in the hydrophobic core of bilayers. Here, the results of a quantitative analysis of cross-relaxation in a lipid 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC)/water mixture are reported. Coherences were selected via application of pulsed magnetic field gradients. This technique shortens acquisition times of NOESY spectra to 20 min and reduces t (1)-spectral noise, enabling detection of weak crosspeaks, like those between water and lipids, with higher precision than with non-gradient NOESY methods. The analysis showed that water molecules interact almost exclusively with sites of the lipid-water interface, including choline, phosphate, glycerol, and carbonyl groups. The lifetime of lipid-water associations is rather short, on the order of 100 ps, at least one order of magnitude shorter than the lifetime of lipid-lipid associations. The distribution of water molecules over the lipid bilayer was measured at identical water content by neutron diffraction. Water molecules penetrate deep into the interfacial region of bilayers but water concentration in the hydrophobic core is below the detection limit of one water molecule per lipid, in excellent agreement with the cross-relaxation data.

NMR analysis of duplex d(CGCGATCGCG)2 modified by Λ- and Δ-[Ru(bpy)2(m-GHK)]Cl2 and DNA photocleavage study

The interaction of the diastereomeric complexes Lambda-[Ru(bpy)2(m-GHK)]Cl2 and Delta-[Ru(bpy)2(m-GHK)]Cl2 (bpy is 2,2'-bipyridine, GHK is glycine-L-histidine-L-lysine) with the deoxynucleotide duplex d(5'-CGCGATCGCG)2 was studied by means of 1H NMR spectroscopy. At a Delta-isomer to DNA ratio of 1:1, significant shifts for the metal complex are observed, whereas there is negligible effect on the oligonucleotide protons and only one intermolecular nuclear Overhauser effect (NOE) is present at the 2D nuclear Overhauser enhancement spectroscopy spectrum. The 1Eta NMR spectrum at ratio 2:1 is characterized by a slight shift for the Delta-isomer's bpy aromatic protons as well as significant shifts for the decanucleotide G4 H1' and Eta2'', A5 H2, G10 H1', T6 NH and G2 NH protons. Furthermore, at ratio 2:1, 11 intermolecular NOEs are observed. The majority of the NOEs involve the sugar Eta2' and Eta2'' protons sited in the major groove of the decanucleotide. Increasing the Delta-isomer to d(CGCGATCGCG)2 ratio to 5:1 results in noteworthy spectral changes. The Delta-isomer's proton shifts are reduced, whereas significant shifts are observed for the decanucleotide protons, especially the sugar protons, as well as for the exchangeable protons. Interaction is characterized by the presence of only one intermolecular NOE. Furthermore, there is significant broadening of the imino proton signals as the ratio of the Delta-isomer to DNuAlpha increases, which is attributed to the opening of the two strands of the duplex. The Lambda-isomer, on the other hand, approaches the minor groove of the oligonucleotide and interacts only weakly, possibly by electrostatic interactions. Photocleavage studies were also conducted with the plasmid pUC19 and a 158-bp restriction fragment, showing that both diastereomers cleave DNA with similar efficiency, attacking mainly the guanines of the sequence probably by generating active oxygen species.

Solution Structure of the Human Immunodeficiency Virus Type 1 p6 Protein

The human immunodeficiency virus type 1 p6 protein represents a docking site for several cellular and viral binding factors and fulfills major roles in the formation of infectious viruses. To date, however, the structure of this 52-amino acid protein, by far the smallest lentiviral protein known, either in its mature form as free p6 or as the C-terminal part of the Pr55 Gag polyprotein has not been unraveled. We have explored the high resolution structure and folding of p6 by CD and NMR spectroscopy. Under membranous solution conditions, p6 can adopt a helix-flexible helix structure; a short helix-1 (amino acids 14-18) is connected to a pronounced helix-2 (amino acids 33-44) by a flexible hinge region. Thus, p6 can be subdivided into two distinct structural and functional domains; helix-2 perfectly defines the region that binds to the virus budding factor AIP-1/ALIX, indicating that this structure is required for interaction with the endosomal sorting complex required for transport. The PTAP motif at the N terminus, comprising the primary late assembly domain, which is crucial for interaction with another cellular budding factor, Tsg101, does not exhibit secondary structure. However, the adjacent helix-1 may play an indirect role in the specific complex formation between p6 and the binding groove in Tsg101. Moreover, binding studies by NMR demonstrate that helix-2, which also comprises the LXXLF motif required for incorporation of the human immunodeficiency virus type 1 accessory protein Vpr into budding virions, specifically interacts with the Vpr binding region, indicating that under the specific solution conditions used for structure analysis, p6 adopted a functional conformation.

Characterization by NMR and molecular modeling of the binding of polyisoprenols and polyisoprenyl recognition sequence peptides: 3D structure of the complexes reveals sites of specific interactions.

The objective of these studies was to test the hypothesis that proteins that contain potential polyisoprenyl recognition sequences (PIRSs) in their transmembrane-spanning domain can bind to the polyisoprenyl (PI) glycosyl carrier lipids undecaprenyl phosphate (C55-P) and dolichyl phosphate (C95-P). A number of prokaryotic and eukaryotic glycosyltransferases that utilize PI coenzymes contain a conserved PIRS postulated to be the active PI binding domain. To study this problem, we first determined the 3D structure of a PIRS peptide, NeuE, by homonuclear 2D 1H-nuclear magnetic resonance (NMR) spectroscopy. Experimentally generated distance constraints derived from nuclear Overhauser enhancement and torsion angle constraints derived from coupling constants were used for restrained molecular dynamics and energy minimization calculations. Molecular models of the NeuE peptide were built based on calculations of energy minimization using the DGII program NMRchitect. 3D models of dolichol (C95) and C95-P were built based on our 2D 1H-NMR nuclear Overhauser enhancement spectroscopy (NOESY) results and refined by energy minimization with respect to all atoms using the AMBER (assisted modeling with energy refinements) force field. Our energy minimization studies were carried out on a conformational model of dolichol that was originally derived from small-angle X-ray scattering and molecular mechanics methods. These results revealed that the PIs are conformationally nearly identical tripartite molecules, with their three domains arranged in a coiled, helical structure. Analyses of the intermolecular cross-peaks in the 2D NOESY spectra of PIRS peptides in the presence of PIs confirmed a highly specific interaction and identified key contact amino acids in the NeuE peptide that constituted a binding motif for interacting with the PIs. These studies also showed that subtle conformational changes occurred within both the PIs and the NeuE peptide after binding. 3D structures of the resulting molecular complexes revealed that each PI could bind more than one PIRS peptide. These studies thus represent the first evidence for a direct physical interaction between specific contact amino acids in the PIRS peptides and the PIs and supports the hypothesis of a bifunctional role for the PIs. The central idea is that these superlipids may serve as a structural scaffold to organize and stabilize in functional domains PIRS-containing proteins within multiglycosyltransferase complexes that participate in biosynthetic and translocation processes.

The Solution Structure of Human Hepcidin, a Peptide Hormone with Antimicrobial Activity That Is Involved in Iron Uptake and Hereditary Hemochromatosis

The antibacterial and antifungal peptide hepcidin (LEAP-1) is expressed in the liver. This circulating peptide has recently been found to also act as a signaling molecule in iron metabolism. As such, it plays an important role in hereditary hemochromatosis, a serious iron overload disease. In this study, we report the solution structures of the hepcidin-20 and -25 amino acid peptides determined by standard two-dimensional (1)H NMR spectroscopy. These small cysteine-rich peptides form a distorted beta-sheet with an unusual vicinal disulfide bridge found at the turn of the hairpin, which is probably of functional significance. Both peptides exhibit an overall amphipathic structure with six of the eight Cys involved in maintaining interstrand connectivity. Hepcidin-25 assumes major and minor conformations centered about the Pro residue near the N-terminal end. Further NMR diffusion studies indicate that hepcidin-20 exists as a monomer in solution, whereas hepcidin-25 readily aggregates, a property that may contribute to the different activities of the two peptides. The nuclear Overhauser enhancement spectroscopy spectra of the hepcidin-25 aggregates indicate an interface for peptide interactions that again involves the first five residues from the N-terminal end.

Sequencing of N‐vinyl‐2‐pyrrolidone/glycidyl methacrylate copolymers by one‐dimensional and two‐dimensional nuclear magnetic resonance spectroscopy

AbstractCopolymers of N‐vinyl‐2‐pyrrolidone (V) and glycidyl methacrylate (G) monomers of different compositions were prepared by free‐radical solution polymerization. The copolymer composition of these copolymers was determined with 1H‐NMR spectra. The reactivity ratios calculated from the Kelen–Tudos and nonlinear least‐square error‐in‐variable methods were rV = 0.03 ± 0.01 and rG = 5.05 ± 0.84 and rV = 0.02 and rG = 4.72, respectively. The triad sequence distribution in terms of V and G centered triads was determined from 13C{1H}‐NMR spectroscopy. The complete spectral assignment of 13C{1H}‐ and 1H‐NMR spectra was performed with the help of distortionless enhancement by polarization transfer and two‐dimensional 13C–1H heteronuclear single quantum coherence. The 1H–1H couplings were explained with total correlation spectroscopy and nuclear Overhauser enhancement spectroscopy spectra. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 50–60, 2002; DOI 10.1002/app.10186

Determination of a high precision structure of a novel protein, Linum usitatissimum trypsin inhibitor (LUTI), using computer-aided assignment of NOESY cross-peaks

The solution structure of a novel 69 residue proteinase inhibitor, Linum usitatissimum trypsin inhibitor (LUTI), was determined using a method based on computer aided assignment of nuclear Overhauser enhancement spectroscopy (NOESY) data. The approach applied uses the program NOAH/DYANA for automatic assignment of NOESY cross-peaks. Calculations were carried out using two unassigned NOESY peak lists and a set of determined dihedral angle restraints. In addition, hydrogen bonds involving amide protons were identified during calculations using geometrical criteria and values of HN temperature coefficients. Stereospecific assignment of β-methylene protons was carried out using a standard procedure based on nuclear Overhauser enhancement intensities and 3Jαβcoupling constants. Further stereospecific assignment of methylene protons and diastereotopic methyl groups were established upon structure-based method available in the program GLOMSA and chemical shift calculations. The applied algorithm allowed us to assign 1968 out of 2164 peaks (91 %) derived from NOESY spectra recorded in H2O and 2H2O. The final experimental data input consisted of 1609 interproton distance restraints, 88 restraints for 44 hydrogen bonds, 63 torsion angle restraints and 32 stereospecifically assigned methylene proton pairs and methyl groups. The algorithm allowed the calculation of a high precision protein structure without the laborious manual assignment of NOESY cross-peaks. For the 20 best conformers selected out of 40 refined ones in the program CNS, the calculated average pairwise rmsd values for residues 3 to 69 were 0.38 Å (backbone atoms) and 1.02 Å (all heavy atoms). The three-dimensional LUTI structure consists of a mixed parallel and antiparallel β-sheet, a single α-helix and shows the fold of the potato 1 family of proteinase inhibitors. Compared to known structures of the family, LUTI contains Arg and Trp residues at positions P6′ and P8′, respectively, instead of two Arg residues, involved in the proteinase binding loop stabilization. A consequence of the Arg→Trp substitution at P8′ is a slightly more compact conformation of the loop relative to the protein core.

Structure of Tuberoinfundibular Peptide of 39 Residues

Structure of Tuberoinfundibular Peptide of 39 Residues

Micellization of sodium dodecyl sulfonate and Triton X-100 in polyacrylamide water solution studied by 1 H NMR relaxation and two-dimensional nuclear overhauser enhancement spectroscopy

Micellization of Triton X-100 (TX-100) and sodium dodecyl sulfonate (SDSN) in the presence of partially hydrolyzed polyacrylamide (PAAM) was studied by 1H NMR spin–spin and spin–lattice relaxation. Relaxation experiment results show that TX-100 behaves differently from SDSN in micellization in the presence of PAAM. PAAM causes a decrease in the critical micellar concentration of SDSN, while it has no influence on the critical micellar concentration of TX-100. The lack of cross peaks between protons of PAAM and those of TX-100 and SDSN in the 2D nuclear Overhauser enhancement spectroscopy (NOESY) spectra confirms self-aggregation of TX-100 and SDSN in the presence of PAAM. The identity of each of the corresponding interproton distances of TX-100 with and without the addition of PAAM further confirms the formation of normal TX-100 micelles in the presence of PAAM. Besides, the distances between protons on the hydrophilic and hydrophobic chains in TX-100 micelles, calculated from the 2D NOESY spectra, are remarkably shorter than those for an extended hydrophilic poly(oxyethylene) chain. This implies that the hydrophilic chain is curled upon micellization.

Solution Structure of Porcine Delta Sleep-inducing Peptide Immunoreactive Peptide A Homolog of the ShortsightedGene Product

The 77-residue delta sleep-inducing peptide immunoreactive peptide (DIP) is a close homolog of the Drosophila melanogaster shortsighted gene product. Porcine DIP (pDIP) and a peptide containing a leucine zipper-related partial sequence of pDIP, pDIP(9-46), was synthesized and studied by circular dichroism and nuclear magnetic resonance spectroscopy in combination with molecular dynamics calculations. Ultracentrifugation, size exclusion chromatography, and model calculations indicated that pDIP forms a dimer. This was confirmed by the observation of concentration-dependent thermal folding-unfolding transitions. From CD spectroscopy and thermal folding-unfolding transitions of pDIP(9-46), it was concluded that the dimerization of pDIP is a result of interaction between helical structures localized in the leucine zipper motif. The three-dimensional structure of the protein was determined with a modified simulated annealing protocol using experimental data derived from nuclear magnetic resonance spectra and a modeling approach based on an established strategy for coiled coil structures. The left-handed super helical structure of the leucine zipper type sequence resulting from the modeling approach is in agreement with known leucine zipper structures. In addition to the hydrophobic interactions between the amino acids at the heptade positions a and d, the structure of pDIP is stabilized by the formation of interhelical i to i' + 5 salt bridges. This result was confirmed by the pH dependence of the thermal-folding transitions. In addition to the amphipatic helix of the leucine zipper, a second helix is formed in the NH2-terminal part of pDIP. This helix exhibits more 310-helix character and is less stable than the leucine zipper helix. For the COOH-terminal region of pDIP no elements of regular secondary structure were observed.

Solution structure of neuropeptide tyrosine 13-36, a Y2 receptor agonist, as determined by NMR.

The three-dimensional structure of neuropeptide tyrosine (NPY) 13-36, a specific Y2 receptor agonist, has been investigated by two-dimensional 1H-NMR spectroscopy in solution. Analysis of the double-quantum-filtered correlation spectroscopy (DQFCOSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY) spectra provided a complete assignment of the proton signals. The interproton connectivities observed in the NOESY spectra comprised 166 intraresidue and 95 interresidue distance ranges which were used as constraints for molecular modeling by distance geometry, simulated annealing and energy minimization. The optimal structures are characterized by a helical C-terminal fragment Leu30-Tyr36 and a wide loop from Leu17 to Ser22. The structure of NPY 13-36 is analogous to the structure of NPY under the same solvent conditions. Comparison with other reported Y2 agonists suggests that the helical Leu30-Tyr36 fragment is the most critical for activity.

A two-dimensional NMR study of bovine Cu, Co superoxide dismutase. Further assignments in the region surrounding the active site.

Bovine Cu, Co superoxide dismutase has been investigated by two-dimensional NMR with regard to the resonances arising from protons that surround the copper site. These protons have magnetic properties that are intermediate between those belonging to residues coordinated to the paramagnetic metal center and the diamagnetic residues. Nuclear Overhauser enhancement spectroscopy spectra have been recorded by using a new procedure and with different mixing times and different spectral widths, in order to observed dipolar connectivities between isotropically shifted and diamagnetic resonances. Scalar-correlated spectra were obtained with both correlation spectroscopy and total-correlation spectroscopy experiments. The original X-ray coordinates of the Cu, Co enzyme were used in order to obtain the appropriate interproton distances. The data allowed us to assign more than 20 new resonances to protons which cover a wide region around the copper ion, including two protons belonging to the catalytically important Arg141 residue. The results represent significant progress in the effort to elucidate the three-dimensional features of the region surrounding the active site of Cu, Zn superoxide dismutase in solution and a tool for a deeper investigation of the reaction mechanism of the enzyme.