Solution conformation of a synthetic fragment of human pituitary growth hormone. Two-dimensional NMR of an alpha-helical dimer

Circular dichroism and two-dimensional NMR spectra indicate that a peptide fragment consisting of the first 28 residues from the N-terminus of human growth hormone (hGH 1-28) has considerable alpha-helical structure. The peptide, (1) H-Phe-Pro-Thr-Ile-Pro-Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-Met-Leu-Arg-Ala-Hi s-Arg- Leu-His-Gln-Leu-Ala-Phe-Asp-Thr-Tyr-OH (28), was synthesized on an automated peptide synthesizer using the Merrifield solid-phase method. The peptide can be modeled as an amphiphilic helix, and the unusual stability of the alpha-helix in aqueous solution is suggested to be attributable to formation of a dimer of alpha-helices. Most of the 1H NMR signals were assigned through pure absorption phase COSY/NOESY and single- and double-relay COSY 2D NMR spectra by using the sequential assignment methodology. The NOEs were large and negative, suggesting that the peptide was not a random coil and that it existed in solution primarily as a large, fairly rigid macromolecule, consistent with the dimer structure. A network of N alpha Hi-N alpha Hi+1 NOESY crosspeaks is observed from residues 13 to 18 as are several other crosspeaks which indicate that the peptide has considerable alpha-helical structure between residues 8 and 24. In addition, gel filtration of the peptide is consistent with a dimer structure, presumably involving packing of the two hydrophobic faces of the amphiphilic alpha-helices.     

Eosinophil peroxidase oxidation of thiocyanate. Characterization of major reaction products and a potential sulfhydryl-targeted cytotoxicity system

Although the pseudohalide thiocyanate (SCN(-)) is the preferred substrate for eosinophil peroxidase (EPO) in fluids of physiologic halide composition, the product(s) of this reaction have not been directly identified, and mechanisms underlying their cytotoxic potential are poorly characterized. We used nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry, and quantitative chemical analysis to identify the principal reaction products of both the EPO/SCN(-)/H(2)O(2) system and activated eosinophils as roughly equimolar amounts of OSCN(-) (hypothiocyanite) and OCN(-) (cyanate). Red blood cells exposed to increasing concentrations of OSCN(-)/OCN(-) are first depleted of glutathione, after which glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase then ATPases undergo sulfhydryl (SH) reductant-reversible inactivation before lysing. OSCN(-)/OCN(-) inactivates red blood cell membrane ATPases 10-1000 times more potently than do HOCl, HOBr, and H(2)O(2). Exposure of glutathione S-transferase to [(14)C]OSCN(-)/OCN(-) causes SH reductant-reversible disulfide bonding and covalent isotope labeling. We propose that EPO/SCN(-)/H(2)O(2) reaction products comprise a potential SH-targeted cytotoxic system that functions in striking contrast to HOCl, the highly but relatively indiscriminantly reactive product of the neutrophil myeloperoxidase system.     

Metabolomic analysis using optimized NMR and statistical methods

NMR-based metabolomics requires robust automated methodologies, and the accuracy of NMR-based metabolomics data is greatly influenced by the reproducibility of data acquisition and processing methods. Effective water resonance signal suppression and reproducible spectral phasing and baseline traces across series of related samples are crucial for statistical analysis. We assess robustness, repeatability, sensitivity, selectivity, and practicality of commonly used solvent peak suppression methods in the NMR analysis of biofluids with respect to the automated processing of the NMR spectra and the impact of pulse sequence and data processing methods on the sensitivity of pattern recognition and statistical analysis of the metabolite profiles. We introduce two modifications to the excitation sculpting pulse sequence whereby the excitation solvent suppression pulse cascade is preceded by low-power water resonance presaturation pulses during the relaxation delay. Our analysis indicates that combining water presaturation with excitation sculpting water suppression delivers the most reproducible and information-rich NMR spectra of biofluids.     

Evaluating the potential of fluorinated tyrosines as spectroscopic probes of local protein environments: a UV resonance Raman study

Ultraviolet resonance Raman (UVRR) studies designed to test the utility of fluorinated tyrosines as spectroscopic probes of the local environment are presented. Specifically, resonance Raman spectra of 2-fluoro-L-tyrosine and 3-fluoro-L-tyrosine (3-Y(f)) obtained with 229 nm excitation are reported. In contrast to the modest environmental dependence of the tyrosine resonance Raman spectrum, the spectrum of 3-Y(f) is found to be extremely dependent on the hydrogen bonding strength of the surrounding environment. Preliminary ab initio studies suggest that this behavior is due to normal modes having dominant contributions from the C-OH and C-F internal coordinates. Hydrogen bonding to the solvent perturbs the internal coordinate energetics and/or couplings, thereby altering the character of the normal modes and the corresponding transition frequencies and/or intensities. In addition to the solvent studies, 3-Y(f) is site specifically incorporated into the influenza hemagglutinin (HA) 100-107 peptide which binds to the Fv fragment of the 17/9 anti-HA(98-108) peptide antibody. These studies demonstrate that the spectrum of 3-Y(f) can be monitored in the presence of native tyrosine. In summary, the studies presented here demonstrate that 3-Y(f) holds exceptional promise as a probe of the protein environment.     

Effect of distortions in the deoxyribose phosphate backbone conformation of duplex oligodeoxyribonucleotide dodecamers containing GT, GG, GA, AC, and GU base-pair mismatches on 31P NMR spectra

We have previously suggested that variations in the 31P chemical shifts of individual phosphates in duplex oligonucleotides are attributable to torsional angle changes in the deoxyribose phosphate backbone. This hypothesis is not directly supported by analysis of the 1H/31P two-dimensional J-resolved spectra of a number of mismatch dodecamer oligonucleotide duplexes including the following sequences: d-(CGTGAATTCGCG), d(CGUGAATTCGCG), d(CGGGAATTCGCG), d(CGAGAATTCGCG), and d(CGCGAATTCACG). The 31P NMR signals of the dodecamer mismatch duplexes were assigned by 2D 1H/31P pure absorption phase constant time (PAC) heteronuclear correlation spectra. From the assigned H3' and H4' signals, the 31P signals of the base-pair mismatch dodecamers were identified. JH3'-P coupling constants for each of the phosphates of the dodecamers were obtained from 1H/31P J-resolved selective proton flip 2D spectra. By use of a modified Karplus relationship, the C4'-C3'-O3'-P torsional angles (epsilon) were obtained. JH3'-P coupling constants were measured for many of the oligonucleotides as a function of temperature. There exists a good linear correlation between 31P chemical shifts and the epsilon torsional angle. This correlation can be further extended to the C3'-O3'-P-O5' torsional angle (zeta) by using a linear relationship between epsilon and zeta obtained from crystal structure studies. The 31P chemical shifts follow the general observation that the more internally the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. In addition, 31P chemical shifts show sequence- and site-specific variations. Analysis of the backbone torsional angle variations from the coupling constant analysis has provided additional information regarding the origin of these variations in 31P chemical shifts.     

Sequence-dependent variations in the 31P NMR spectra and backbone torsional angles of wild-type and mutant Lac operator fragments

Assignment of the 31P resonances of a series of six sequenced-related tetradecamer DNA duplexes, d(TGTGAGCGCTCACA)2, d(TATGAGCGCTCATA)2, d(TCTGAGCGCTCAGA)2, d(TGTGTGCGCACACA)2, d(TGTGACGCGTCACA)2 and d(CACAGTATACTGTG)2, related to the lac operator DNA sequence was determined either by site-specific 17O labeling of the phosphoryl groups or by two-dimensional 1H-31P pure absorption phase constant time (PAC) heteronuclear correlation spectroscopy. J(H3'-P) coupling constants for each of the phosphates of the tetradecamers were obtained from 1H-31P J-resolved selective proton flip 2D spectra. By use of a modified Karplus relationship the C4'-C3'-O3'-P torsional angles (epsilon) were obtained. Comparison of the 31P chemical shifts and J(H3'-P) coupling constants of these sequences has allowed greater insight into those various factors responsible for 31P chemical shift variations in oligonucleotides and provided an important probe of the sequence-dependent structural variation of the deoxyribose phosphate backbone of DNA in solution. These sequence-specific variations in the conformation of the DNA sugar phosphate backbone of various lac operator DNA sequences can possibly explain the sequence-specific recognition of DNA by DNA binding proteins, as mediated through direct contacts between the phosphates and the protein.     

Assignments of 31P NMR resonances in oligodeoxyribonucleotides: origin of sequence-specific variations in the deoxyribose phosphate backbone conformation and the 31P chemical shifts of double-helical nucleic acids

It is now possible to unambiguously assign all 31P resonances in the 31P NMR spectra of oligonucleotides by either two-dimensional NMR techniques or site-specific 17O labeling of the phosphoryl groups. Assignment of 31P signals in tetradecamer duplexes, (dTGTGAGCGCTCACA)2, (dTAT-GAGCGCTCATA)2, (dTCTGAGCGCTCAGA)2, and (dTGTGTGCGCACACA)2, and the dodecamer duplex d(CGTGAATTCGCG)2 containing one base-pair mismatch, combined with additional assignments in the literature, has allowed an analysis of the origin of the sequence-specific variation in 31P chemical shifts of DNA. The 31P chemical shifts of duplex B-DNA phosphates correlate reasonably well with some aspects of the Dickerson/Calladine sum function for variation in the helical twist of the oligonucleotides. Correlations between experimentally measured P-O and C-O torsional angles and results from molecular mechanics energy minimization calculations show that these results are consistent with the hypothesis that sequence-specific variations in 31P chemical shifts are attributable to sequence-specific changes in the deoxyribose phosphate backbone. The major structural variation responsible for these 31P shift perturbations appears to be P-O and C-O backbone torsional angles which respond to changes in the local helical structure. Furthermore, 31P chemical shifts and JH3'-P coupling constants both indicate that these backbone torsional angle variations are more permissive at the ends of the double helix than in the middle. Thus 31P NMR spectroscopy and molecular mechanics energy minimization calculations appear to be able to support sequence-specific structural variations along the backbone of the DNA in solution.     

Two-dimensional 1H and 31P NMR spectra and restrained molecular dynamics structure of an extrahelical adenosine tridecamer oligodeoxyribonucleotide duplex

Assignment of the 1H and 31P NMR spectra of an extrahelical adenosine tridecamer oligodeoxyribonucleotide duplex, d(CGCAGAATTCGCG)2, has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. The downfield 31P resonance previously noted by Patel et al. (1982) has been assigned by both 17O labeling of the phosphate as well as a pure absorption phase constant-time heteronuclear 31P-1H correlated spectrum and has been associated with the phosphate on the 3' side of the extrahelical adenosine. JH3'-P coupling constants for each of the phosphates of the tridecamer were obtained from the 1H-31P J-resolved selective proton-flip 2D spectrum. By use of a modified Karplus relationship the C4-C3'-O3-P torsional angles (epsilon) were obtained. There exists a good linear correlation between 31P chemical shifts and the epsilon torsional angle. The 31P chemical shifts and epsilon torsional angles follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. Because the extrahelical adenosine significantly distorts the deoxyribose phosphate backbone conformation even several bases distant from the extrahelical adenosine, 31P chemical shifts show complex site- and sequence-specific variations. Modeling and NOESY distance-restrained energy minimization and restrained molecular dynamics suggest that the extrahelical adenosine stacks into the duplex. However, a minor conformation is also observed in the 1H NMR, which could be associated with a structure in which the extrahelical adenosine loops out into solution.