Determination of local conformational stability in fragment 96-133 of bovine growth hormone by high-resolution H NMR spectroscopy

The specific assignment of resonances in the 400-MHz nuclear magnetic resonance (NMR) spectrum of fragment 96-133 (AII) of bovine growth hormone (bSt) is described. Assignments have been made with homonuclear two-dimensional techniques, in particular that of sequential resonance assignment. Complete assignments were possible for the spin systems of 16 residues out of a total of 38 and partial assignments for another 5. Assignment of resonances to either residue type or a class of residue was possible for a number of other spin systems. Analysis of the type of nuclear Overhauser effect (NOE) indicates that segments 96-110 and 130-133 are nonregular stable structures and that the segment 111-127, which putatively spans the alpha-helix, is not sufficiently stable to generate NOEs.     

Identification by 1H NMR spectroscopy of flexible C-terminal extensions in bovine lens alpha-crystallin.

Two-dimensional 1H NMR spectroscopy of bovine eye lens alpha-crystallin and its isolated alpha A and alpha B subunits reveals that these aggregates have short and very flexible C-terminal extensions of eight (alpha A) and ten (alpha B) amino acids which adopt little preferred conformation in solution. Total alpha-crystallin forms a tighter aggregate than the isolated alpha A and alpha B subunit aggregates. Our results are consistent with a micelle model for alpha-crystallin quaternary structure. The presence of terminal extensions is a general feature of those crystallins, alpha and beta, which form aggregates.     

Preferred conformational state of the N-terminus section of a bovine growth hormone fragment (residues 96-133) in water is an omega loop

The solution structure of a 38-amino-acid-residue, biologically active fragment of bovine growth hormone (bGH96-133) was investigated with a combined nuclear magnetic resonance (NMR) and computer modeling approach. With the distance geometry program DISGEO and distance constraints derived from nuclear Overhauser enhancement (NOE) experiments, it was found that residues Ser-100 to Tyr-110 circumscribe and omega-loop, a recently categorized feature of nonregular secondary protein structure.     

The three-dimensional structure of bovine calcium ion-bound osteocalcin using 1H NMR spectroscopy

Structural information on osteocalcin or other noncollagenous bone proteins is very limited. We have solved the three-dimensional structure of calcium bound osteocalcin using (1)H 2D NMR techniques and proposed a mechanism for mineral binding. The protons in the 49 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. Distance constraints, dihedral angle constraints, hydrogen bonds, and (1)H and (13)C chemical shifts were all used to calculate a family of 13 structures. The tertiary structure of the protein consisted of an unstructured N terminus and a C-terminal loop (residues 16-49) formed by long-range hydrophobic interactions. Elements of secondary structure within residues 16-49 include type III turns (residues 20-25) and two alpha-helical regions (residues 27-35 and 41-44). The three Gla residues project from the same face of the helical turns and are surface exposed. The genetic algorithm-molecular dynamics simulation approach was used to place three calcium atoms on the NMR-derived structure. One calcium atom was coordinated by three side chain oxygen atoms, two from Asp30, and one from Gla24. The second calcium atom was coordinated to four oxygen atoms, two from the side chain in Gla 24, and two from the side chain of Gla 21. The third calcium atom was coordinated to two oxygen atoms of the side chain of Gla17. The best correlation of the distances between the uncoordinated Gla oxygen atoms is with the intercalcium distance of 9.43 A in hydroxyapatite. The structure may provide further insight into the function of osteocalcin.     

Characterization of an artificial dimer of ribonuclease H using 1H NMR spectroscopy

Summary The protein fusion technique was applied in the synthesis of an artificial dimer of ribonuclease H (305 residues). ¹H NMR spectroscopy was used to analyze the structure of this dimer. Spectral profiles and pK_a values of the histidine residues obtained using ¹H NMR indicate that the dimer retains the secondary and tertiary structures of the intact monomer. Selective spin-lattice relaxation measurements suggest that the two monomeric units in the dimer are in tight contact. Furthermore, the 2D ¹H NMR and paramagnetic relaxation filter results show that the two monomers bind together through interactions between the N- and C-terminal sites of the linked regions.     

Metabolic profiling of serum from gadolinium chloride-treated rats by 1H NMR spectroscopy

Metabolic profiling of serum from gadolinium chloride (GdCl(3), 10 and 50 mg/kg body weight, intraperitoneal [i.p.])-treated rats was investigated by the NMR spectroscopic-based metabonomic strategy. Serum samples were collected at 48, 96, and 168h postdose (p.d.) after exposure to GdCl(3). (1)H NMR spectra of serum were analyzed by pattern recognition using principal components analysis. The studies showed that there was a dose-related biochemical effect of GdCl(3) treatment on the levels of a range of low-molecular weight compounds in serum. The liver damage induced by GdCl(3) was characterized by the elevation of lactate, pyruvate, and creatine as well as the decrease of branched-chain amino acids (valine and isoleucine), alanine, glucose, and trimethylamine-N-oxide concentration in serum samples. The biochemical effects of GdCl(3) in rats could be consulted when evaluating the biochemical profile of gadolinium-containing compounds that are being developed for nuclear magnetic resonance imaging.     

Sequential resonance assignments in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy

A sequential assignment procedure is outlined, based on two-dimensional NOE ( NOESY ) and two-dimensional J-correlated spectroscopy ( COSY ), for assigning the nonexchangeable proton resonances in NMR spectra of oligonucleotides. As presented here the method is generally applicable to right-handed helical oligonucleotides of intermediate size. We applied it to a lac operator DNA fragment consisting of d( TGAGCGG ) and d( CCGCTCA ) and obtained complete assignments for the adenine H8, guanine H8, cytosine H6 and H5, thymine H6 and 5-methyl, and the deoxyribose H1', H2', H2", H3', and H4' resonances, as well as some H5', H5" (pairwise) assignments. These assignments are required for the analysis of two-dimensional NOE and J-coupling data in terms of the solution structure of oligonucleotides.     

Conformation of an octapeptide fragment (2-9) of kaliocin-1 in DMSO-d6 by 1H NMR and restrained molecular dynamics

Kaliocin-1, a 31-residue synthetic peptide (FFSASCVPGADKGQFPNLCRLCA GTGENKCA), which has shown the antimicrobial activity forms the 152-182 fragment of human lactoferrin (HLf). As the octapeptide FSASCVPG forms the 2-9 fragment of kaliocin-1, in the present study, its conformation in dimethyl sulfoxide-d6 (DMSO-d6) has been determined using two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy as well as restrained molecular dynamics. Sequence specific assignments of all the 1H resonances have been carried out using 2D correlation experiments (2D DQF-COSY, TOCSY and ROESY). In dimethyl sulfoxide-d6 at 25 degrees C, the octapeptide adopts a predominantly extended backbone conformation. The calculated structure resembles closely with the reported structure of the corresponding fragment of HLf. The peptide also has sequence and structural similarity with the corresponding fragments of lactoferrins from other organisms.     

1H NMR relaxometric characterization of bovine lactoferrin

Lactoferrin (Lf) is a mammalian iron binding protein present in external secretions and in polymorphonuclear leukocytes. Its role in host defense mechanisms related to the non-immune defense system has been definitively established. Lf has two identical iron-binding sites, far from each other (44.3 A) and magnetically non-interacting. Fe(III) ions are six-coordinated, with four donor atoms provided by protein sidechains (two Tyr, one His, one Asp) and two oxygen atoms from a bridged HCO(3)(-). This set of ligands provides an ideal coordination scheme for stable and reversible iron binding. Nuclear magnetic relaxation dispersion (NMRD) profiles of Lf are consistent with a closest distance for a single water hydrogen atom of 3.1 A. By looking at the X-ray structure of Lf (PDB ID code: 1BLF) we can locate two water oxygens at 3.95 and 4.27 A from each Fe(III), respectively. Temperature dependence data suggest that an important contribution to the overall paramagnetic contribution to the solvent water relaxation rate arises from one or more second sphere water molecules in slow exchange with the bulk. A decreasing value of the exchange rate is obtained, ranging from 1.2 to 0.7 micros in the observed temperature range (25-65 degrees C), with an activation enthalpy of 7.3+/-0.8 kJ mol(-1). The low exchange rate obtained from NMRD data can be explained by the observation that both water molecules are bound to several polar groups of the protein backbone and side chains. By increasing the pH from 6.5 to 12 two distinct titrations are observed, consistent with sequential removal of both water molecules.     

Secondary structure determination of human beta-endorphin by 1H NMR spectroscopy

The 1H NMR spectra of human beta-endorphin indicate that the peptide exists in random-coil form in aqueous solution but becomes helical in mixed solvent. Thermal denaturation NMR experiments show that in water there is no transition between 24 and 75 degrees C, while a slow noncooperative thermal unfolding is observed in a 60% methanol-40% water mixed solvent in the same temperature range. These findings are consistent with circular dichroism studies by other workers concluding that beta-endorphin is a random coil in water but that it forms 50% alpha-helix or more in mixed solvents. The peptide in the mixed water-methanol solvent was further studied by correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments. These allow a complete set of assignments to be made and establish two distinct stretches over which the solvent induces formation of alpha-helices: the first occurs between Tyr-1 and Thr-12 and the second between Leu-14 and extending to Lys-28. There is evidence that the latter is capped by a turn occurring between Lys-28 and Glu-31. These helices form at the enkephalin receptor binding site, which is at the amino terminus, and at the morphine receptor binding site, located at the carboxyl terminus [Li, C. H. (1982) Cell (Cambridge, Mass.) 31, 504-505]. Our findings suggest that these two receptors may specifically recognize alpha-helices.     

The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy

The structures of the entire population of sialylated asparagine-linked oligosaccharides present on bovine fetuin were elucidated. Asparagine-linked oligosaccharides were released from fetuin with N-glycanase, radiolabeled by reduction with NaB[3H]4, and fractionated by anion-exchange high performance liquid chromatography (HPLC), ion-suppression amine adsorption HPLC, and concanavalin A affinity chromatography. The 3H-labeled oligosaccharide fractions obtained were analyzed by 500-MHz 1H nuclear magnetic resonance spectroscopy, revealing the presence of 23 distinct oligosaccharide structures. These oligosaccharides differed in extent of sialylation (3% mono-, 35% di-, 54% tri-, and 8% tetrasialylated), number of peripheral branches (17% di- and 83% tribranched), linkage (alpha 2,3 versus alpha 2,6) and location of sialic acid moieties, and linkage (beta 1,4 versus beta 1,3) of galactose residues. This represents the first time that the asparagine-linked oligosaccharides of fetuin have been successfully fractionated and characterized as sialylated species. The sialylated oligosaccharides derived from fetuin were also used to further define the specificities of the lectins leukoagglutinating phytohemagglutinin and Ricinus communis agglutinin I. The behavior of these oligosaccharides during lectin affinity HPLC further establishes the structural features which predominate in the interaction of oligosaccharides with leukoagglutinating phytohemagglutinin and R. communis agglutinin I.     

Structure determination of UL49.5 transmembrane protein from bovine herpesvirus 1 by NMR spectroscopy and molecular dynamics

The transporter associated with antigen processing (TAP) directly participates in the immune response as a key component of the cytosolic peptide to major histocompatibility complex (MHC) class I protein loading machinery. This makes TAP an important target for viruses avoiding recognition by CD8+ T lymphocytes. Its activity can be suppressed by the UL49.5 protein produced by bovine herpesvirus 1, although the mechanism of this inhibition has not been understood so far.Therefore, the main goal of our study was to investigate the 3D structure of bovine herpesvirus 1 - encoded UL49.5 protein. The final structure of the inhibitor was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane mimetic environments. In NMR studies, UL49.5 was represented by two fragments: the extracellular region (residues 1–35) and the transmembrane-intracellular fragment (residues 36–75), displaying various functions during viral invasion. After the empirical structure determination, a molecular docking procedure was used to predict the complex of UL49.5 with the TAP heterodimer.Our results revealed that UL49.5 adopted a highly flexible membrane-proximal helical structure in the extracellular part. In the transmembrane region, we observed two short α-helices. Furthermore, the cytoplasmic part had an unordered structure. Finally, we propose three different orientations of UL49.5 in the complex with TAP. Our studies provide, for the first time, the experimental structural information on UL49.5 and structure-based insight in its mechanism of action which might be helpful in designing new drugs against viral infections.     

Identification of an ordered compact structure within the recombinant bovine fibrinogen alphaC-domain fragment by NMR

The NMR solution structure of the bovine fibrinogen alphaC-domain fragment, including residues Aalpha374-538, reveals a type-I' beta-hairpin, restricted at the base by a C423-C453 disulfide linkage and a short turn preceding C423. Although both faces of the hairpin are formed mainly by hydrophilic residues, one of them is uncharged while the other has a characteristic pattern of charged residues which are highly conserved among vertebrate species. Chemical shift indexing and relaxation data indicate the presence of a collapsed hydrophobic region next to the hairpin that includes approximately 30 residues with slower concerted motion and higher content of nonpolar residues and, according to a previous study (Tsurupa, G., Tsonev, L., and Medved, L. (2002) Biochemistry 41, 6449-6459), may cooperate with the hairpin to form a compact cooperative unit (domain). Structure and relaxation data show that the region between C423 and C453 is populated by both random coil and beta-structure, suggesting that the cooperative structure in the isolated alphaC-domain is intrinsically unstable. This observation is in agreement with a very low energy of stabilization of the Aalpha374-538 fragment determined in unfolding experiments. The low stability of the alphaC-domain suggests a possible explanation for the previously observed intra- and intermolecular interactions of these domains in fibrinogen and fibrin.     

113Cd NMR study of bovine prothrombin fragment 1 and factor X

The interaction of Cd2+ with bovine prothrombin fragment 1, prothrombin intermediate 1, factor X, and a modified (Gla-domainless) factor X has been studied with 113Cd NMR. All the 113Cd resonances observed in this study were in the chemical shift range expected for oxygen ligands, suggesting that cadmium is binding at the same sites where calcium binds. Both fragment 1 and factor X displayed two major resonances, one near 10 ppm from 113Cd2+ that did not exchange rapidly with unbound 113Cd2+ (the high-affinity, or H, resonance) and one near -15 ppm from 113Cd2+ that exchanged rapidly with unbound 113Cd2+ (the low-affinity, or L, resonance). The difference between the chemical shift of the H resonance and the chemical shift range of -90 to -125 ppm that has been reported for three other small calcium-binding proteins is postulated to be due to different coordination geometries for monocarboxylate and dicarboxylate ligands; Cd2+ binds to fragment 1 and factor X through the dicarboxylate side chains of gamma-carboxyglutamate (Gla) residues. This allows contribution of only one oxygen per carboxyl group. At least one of the first few 113Cd2+ ions bound to fragment 1 did not appear in the 113Cd NMR spectrum until a total of five 113Cd2+ had been added. This could be due to exchange broadening of initial 113Cd2+ resonances due to sharing of ligands among several sites. Filling all sites would then restrict ligand exchange. Addition of Zn2+ displaced 113Cd2+ from the H resonance sites. Factor X did not display the interactions among ion binding sites proposed for fragment 1.     

Metabotyping of long-lived mice using 1H NMR spectroscopy

Significant advances in understanding aging have been achieved through studying model organisms with extended healthy lifespans. Employing 1H NMR spectroscopy, we characterized the plasma metabolic phenotype (metabotype) of three long-lived murine models: 30% dietary restricted (DR), insulin receptor substrate 1 null (Irs1-/-), and Ames dwarf (Prop1df/df). A panel of metabolic differences were generated for each model relative to their controls, and subsequently, the three long-lived models were compared to one another. Concentrations of mobile very low density lipoproteins, trimethylamine, and choline were significantly decreased in the plasma of all three models. Metabolites including glucose, choline, glycerophosphocholine, and various lipids were significantly reduced, while acetoacetate, d-3-hydroxybutyrate and trimethylamine-N-oxide levels were increased in DR compared to ad libitum fed controls. Plasma lipids and glycerophosphocholine were also decreased in Irs1-/- mice compared to controls, as were methionine and citrate. In contrast, high density lipoproteins and glycerophosphocholine were increased in Ames dwarf mice, as were methionine and citrate. Pairwise comparisons indicated that differences existed between the metabotypes of the different long-lived mice models. Irs1-/- mice, for example, had elevated glucose, acetate, acetone, and creatine but lower methionine relative to DR mice and Ames dwarfs. Our study identified several potential candidate biomarkers directionally altered across all three models that may be predictive of longevity but also identified differences in the metabolic signatures. This comparative approach suggests that the metabolic networks underlying lifespan extension may not be exactly the same for each model of longevity and is consistent with multifactorial control of the aging process.     

Investigation of the solution structure of the human parathyroid hormone fragment (1-34) by 1H NMR spectroscopy, distance geometry, and molecular dynamics calculations

The structure of human parathyroid hormone fragment (1-34) in a solvent mixture of water and trifluoroethanol has been determined by 1H nuclear magnetic resonance spectroscopy and a combination of distance geometry and molecular dynamic simulations. After complete assignment of the 1H signals, the nuclear Overhauser enhancement data imply the existence of two alpha-helices, comprising residues 3-9 and 17-28, joined by a nonstructured region. The absence of any long-range NOEs and the relative magnitudes of the sequential NOEs and the 3J(HNH alpha) values reflect an inherent flexibility within the entire fragment. The final structures refined by molecular dynamics further support the above results and allow discussion of structural-activity relationships.     

An inflammatory arthritis-associated metabolite biomarker pattern revealed by 1H NMR spectroscopy

Rheumatoid arthritis, a debilitating, systemic inflammatory joint disease, is likely accompanied by alterations in circulating metabolites. Here, an 1H NMR spectroscopy-based metabolomics approach was developed to establish a metabolic 'biomarker pattern' in a model of rheumatoid arthritis, the K/BxN transgenic mouse. Sera obtained from arthritic K/BxN mice (N = 15) and a control population (N = 19) having the same genetic background, but lacking the arthritogenic T-cell receptor KRN transgene, were compared by 1H NMR spectroscopy. A unique method was developed by combining technologies such as ultrafiltration to remove proteins from serum samples, quantitative 'targeted profiling' of known metabolites, pseudo-quantitative profiling of unknown resonances, a supervised O-PLS-DA pattern recognition analysis, and a metabolic-pathway based network analysis for interpretation of results. In total, 88 spectral features were profiled (59 metabolites and 28 unknown resonances). A highly significant subset of 18 spectral features (15 known compounds and 3 unknown resonances) was identified (p = 0.00075 using MANOVA) that we term a 'metabolic bioprofile'. We identified metabolites relating to nucleic acid, amino acid, and fatty acid metabolism, as well as lipolysis, reactive oxygen species generation, and methylation. Pathway analysis suggested a shift from metabolites involved in numerous reactions (hub-metabolites) toward intermediates and metabolic endpoints associated with arthritis. The results attest to the metabolic complexity of systemic inflammation and to the power of the experimental approach for identifying a wide variety of disease-associated marker candidates. The diagnostic and prognostic implications of monitoring a spectrum of metabolic events simultaneously using serum samples is discussed with respect to the potential for individualized medicine.     

Human platelet factor 4 monomer-dimer-tetramer equilibria investigated by 1H NMR spectroscopy

As a function of protein concentration, proton NMR spectra of human platelet factor 4 (PF4) differ. Correlation with low-angle laser light scattering data has allowed identification of concentration-dependent NMR spectral changes to PF4 aggregation, with tetramers being the largest aggregates formed. Well-resolved aromatic ring proton NMR resonances were assigned to Tyr-60, His-I, and His-II in monomer, dimer, and tetramer states. Since Tyr-60 3.5 ring proton resonances are well resolved from state to state, estimation of fractional populations in each state was possible. By varying the PF4 concentration, changes in these populations when plotted according to the Hill equation show a bimolecular mechanism of aggregation which proceeds from monomers to tetramers through a dimer intermediate. Equilibrium constants for dimer association (KD) and tetramer association (KT) have been estimated as a function of pH and ionic strength. At pH 4, where KD and KT approach the same value, resonances associated with all three aggregate states are observed. Lowering the pH shifts the equilibrium to the monomer state, while raising the pH shifts the equilibrium to dimer and tetramer states. Analysis of the pH dependence of KD and KT suggests that electrostatic interactions, probably arising from Glu/Asp and Lys/Arg side chains, play a role in the binding process. Increasing the solvent ionic strength stabilizes the tetramer state especially at low pH, suggesting that intersubunit, repulsive electrostatic interactions probably between/among cationic side chains (Lys/Arg) attenuate the aggregation process. Information based primarily on histidine pKa values and photo-CIDNP 1H NMR data suggests that Tyr-60 and His-I, but not His-II, are significantly affected by the aggregation process.     

Solid-state NMR studies of the prion protein H1 fragment.

Conformational changes in the prion protein (PrP) seem to be responsible for prion diseases. We have used conformation-dependent chemical-shift measurements and rotational-resonance distance measurements to analyze the conformation of solid-state peptides lacking long-range order, corresponding to a region of PrP designated H1. This region is predicted to undergo a transformation of secondary structure in generating the infectious form of the protein. Solid-state NMR spectra of specifically 13C-enriched samples of H1, residues 109-122 (MKHMAGAAAAGAVV) of Syrian hamster PrP, have been acquired under cross-polarization and magic-angle spinning conditions. Samples lyophilized from 50% acetonitrile/50% water show chemical shifts characteristic of a beta-sheet conformation in the region corresponding to residues 112-121, whereas samples lyophilized from hexafluoroisopropanol display shifts indicative of alpha-helical secondary structure in the region corresponding to residues 113-117. Complete conversion to the helical conformation was not observed and conversion from alpha-helix back to beta-sheet, as inferred from the solid-state NMR spectra, occurred when samples were exposed to water. Rotational-resonance experiments were performed on seven doubly 13C-labeled H1 samples dried from water. Measured distances suggest that the peptide is in an extended, possibly beta-strand, conformation. These results are consistent with the experimental observation that PrP can exist in different conformational states and with structural predictions based on biological data and theoretical modeling that suggest that H1 may play a key role in the conformational transition involved in the development of prion diseases.