NMR metabolomics profiling of blood plasma mimics shows that medium- and long-chain fatty acids differently release metabolites from human serum albumin.

Metabolite profiling by NMR of body fluids is increasingly used to successfully differentiate patients from healthy individuals. Metabolites and their concentrations are direct reporters of body biochemistry. However, in blood plasma the NMR-detected free-metabolite concentrations are also strongly affected by interactions with the abundant plasma proteins, which have as of yet not been considered much in metabolic profiling. We previously reported that many of the common NMR-detected metabolites in blood plasma bind to human serum albumin (HSA) and many are released by fatty acids present in fatted HSA. HSA is the most abundant plasma protein and main transporter of endogenous and exogenous metabolites. Here, we show by NMR how the two most common fatty acids (FAs) in blood plasma - the long-chain FA, stearate (C18:0) and medium-chain FA, myristate (C14:0) - affect metabolite-HSA interaction. Of the set of 18 common NMR-detected metabolites, many are released by stearate and/or myristate, lactate appearing the most strongly affected. Myristate, but not stearate, reduces HSA-binding of phenylalanine and pyruvate. Citrate signals were NMR invisible in the presence of HSA. Only at high myristate-HSA mole ratios 11:1, is citrate sufficiently released to be detected. Finally, we find that limited dilution of blood-plasma mimics releases HSA-bound metabolites, a finding confirmed in real blood plasma samples. Based on these findings, we provide recommendations for NMR experiments for quantitative metabolite profiling.

Active site substitution A82W improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalized by spin relaxation nuclear magnetic resonance studies.

Cytochrome P450 BM3 from Bacillus megaterium is a monooxygenase with great potential for biotechnological applications. In this paper, we present engineered drug-metabolizing P450 BM3 mutants as a novel tool for regioselective hydroxylation of steroids at position 16ß. In particular, we show that by replacing alanine at position 82 with a tryptophan in P450 BM3 mutants M01 and M11, the selectivity toward 16ß-hydroxylation for both testosterone and norethisterone was strongly increased. The A82W mutation led to a ≤42-fold increase in V(max) for 16ß-hydroxylation of these steroids. Moreover, this mutation improves the coupling efficiency of the enzyme, which might be explained by a more efficient exclusion of water from the active site. The substrate affinity for testosterone increased at least 9-fold in M11 with tryptophan at position 82. A change in the orientation of testosterone in the M11 A82W mutant as compared to the orientation in M11 was observed by T(1) paramagnetic relaxation nuclear magnetic resonance. Testosterone is oriented in M11 with both the A- and D-ring protons closest to the heme iron. Substituting alanine at position 82 with tryptophan results in increased A-ring proton-iron distances, consistent with the relative decrease in the level of A-ring hydroxylation at position 2ß.

1H, 13C and 15N NMR assignments of Duck HBV primer loop of the encapsidation signal epsilon.

The replication of the hepatitis B virus is initiated by binding of the viral reverse transcriptase protein complex to the apical stem loop of the epsilon element to place it next to the primer loop, from which a four nucleotide DNA primer is subsequently synthesized. Here, we present the (1)H/(13)C/(15)N NMR assignments of the bases and sugars of the 37 residues primer loop of Duck HBV epsilon (BMRB-entry 15786).

1H, 13C and 15N NMR assignments of Duck HBV apical stem loop of the epsilon encapsidation signal.

The replication of Hepatitis B virus is initiated by binding of its reverse transcriptase to the apical stem loop and primer loop of epsilon. Here, we present the (1)H/(13)C/(15)N NMR assignments of the bases and sugars of the 29 residues apical stem loop of Duck HBV epsilon.

Prediction of proton chemical shifts in RNA. Their use in structure refinement and validation.

An analysis is presented of experimental versus calculated chemical shifts of the non-exchangeable protons for 28 RNA structures deposited in the Protein Data Bank, covering a wide range of structural building blocks. We have used existing models for ring-current and magnetic-anisotropy contributions to calculate the proton chemical shifts from the structures. Two different parameter sets were tried: (i) parameters derived by Ribas-Prado and Giessner-Prettre (GP set) [(1981) J. Mol. Struct., 76, 81-92.]; (ii) parameters derived by Case [(1995) J. Biomol. NMR, 6, 341-346]. Both sets lead to similar results. The detailed analysis was carried using the GP set. The root-mean-square-deviation between the predicted and observed chemical shifts of the complete database is 0.16 ppm with a Pearson correlation coefficient of 0.79. For protons in the usually well-defined A-helix environment these numbers are, 0.08 ppm and 0.96, respectively. As a result of this good correspondence, a reliable analysis could be made of the structural dependencies of the 1H chemical shifts revealing their physical origin. For example, a down-field shift of either H2' or H3' or both indicates a high-syn/syn chi-angle. In an A-helix it is essentially the 5'-neighbor that affects the chemical shifts of H5, H6 and H8 protons. The H5, H6 and H8 resonances can therefore be assigned in an A-helix on the basis of their observed chemical shifts. In general, the chemical shifts were found to be quite sensitive to structural changes. We therefore propose that a comparison between calculated and observed 1H chemical shifts is a good tool for validation and refinement of structures derived from NOEs and J-couplings.

A novel target recognition revealed by calmodulin in complex with the basic helix--loop--helix transcription factor SEF2-1/E2-2.

Calmodulin is the predominant intracellular receptor for Ca(2+) signals, mediating the regulation of numerous cellular processes. It can inhibit the DNA binding of basic helix--loop--helix transcription factors by a direct interaction of a novel type. To structurally characterize this novel calmodulin-target interaction, we decided to study the complex of calmodulin with a dimeric peptide corresponding to the DNA-binding domains of the dimeric basic helix-loop-helix transcription factor SEF2-1 (SEF2-1mp) using NMR. Here, we report that the stoichiometry of the calmodulin:SEF2-1mp complex is one dimeric peptide binding two calmodulin molecules. We also report the 1H, 13C, and 15N resonance assignments and the secondary structure of calmodulin in this for NMR large (approximately 38 kD) complex, as well as the 1H assignments and secondary structure of SEF2-1mp. In addition, we determined the amide proton exchange rates of calmodulin and measured intermolecular calmodulin:SEF2-1mp and calmodulin:calmodulin NOE contacts. The isotope-filtered experiments show a large number of SEF2-1mp to calmodulin NOE contacts indicating that a tight complex is formed, which is confirmed by an intermolecular calmodulin:calmodulin NOE contact. The secondary structure and amide proton exchange data show that the binding does not occur via the classical wraparound binding mode. Instead, the data indicate that calmodulin interacts with SEF2-1mp in a more open conformation, although the hydrophobic surfaces of the N- and C-terminal domains still form the main interaction sites. Interactions involving charged residues are also identified in agreement with the known relatively high sensitivity of the binding to ionic strength. Finally, the peptide does not form an alpha-helix as in the classical wraparound binding mode.

Solution structure of a DNA three-way junction containing two unpaired thymidine bases. Identification of sequence features that decide conformer selection.

The solution structure of a DNA three-way junction (3H) containing two unpaired thymidine bases at the branch site (3HT2), was determined by NMR. Arms A and B of the 3HT2 form a quasi-continuous stacked helix, which is underwound at the junction and has an increased helical rise. The unstacked arm C forms an acute angle of approximately 55 degrees with the unique arm A. The stacking of the unpaired thymidine bases on arm C resembles the folding of hairpin loops. From this data, combined with the reported stacking behavior of 23 other 3HS2 s, two rules are derived that together correctly reproduce their stacking preference. These rules predict, from the sequence of any 3HS2, its stacking preference. The structure also suggests a plausible mechanism for structure-specific recognition of branched nucleic acids by proteins.

Synthesis of specifically deuterated nucleotides for NMR studies on RNA.

Abstract We propose a strategy for NMR structure determination of RNA based on deuteration and use of specific labeling patterns. This strategy involves the use of NTPs that are deuterated in the ribose ring except for specific positions, e.g. H2', and that are either unlabeled or uniformly labeled in (13)C and (15)N in either the ribose or the base or both. Incorporation of these NTPs into an RNA sequence reduces both resonance line-width and spectral overlap. A limited number of combinations of these differently labeled NTPs in an RNA sequence suffices to obtain all relevant proton resonance assignments and structure parameters necessary for structure determination of larger systems (≫ 50 nucleotides). We describe the in vitro synthesis of the deuterated and/or (13)C/(15)N-labeled NTPs from glucose via separate enzymatic reactions. First, enzymes from the pentose-phosphate pathway efficiently convert glucose into ribose and enzymes from nucleotide biosynthesis and salvage pathways subsequently convert the ribose into nucleosides triphosphates (NTPs). The enzymes from the pentosephosphate pathway are all commercially available; the remaining enzymes have been purified from over-expressing strains. Separate enzymatic reactions were used to convert (2)H(7)- (13)C(6)-glucose into [1',3',4',5',5″-(2)H(5)-1',2',3',4',5',2,4,5,6-(13)C(9)-1,3-(15)N(2)]UTP and (2)H(7)-glucose into [1',3',4',5',5″-(2)H(5)]ATP, [1',3',4',5',5″-(2)H(5)]GTP, and [1',3',4',5',5″-(2)H(5)] CTP. The synthesis yields up to 1 gram of NTPs from 1 gram of glucose, which is about 5 to 10 times as efficient extraction for E. Coli grown on glucose. The synthesis presented here, is a modification of the method described by Tolbert & Williamson (1,2). (1)D and (2)D NMR spectra were acquired to demonstrate the utility of the new labeling patterns. The enzymatically synthesized NTPs were used in the synthesis of a 31-nucleotide RNA derived from the primer binding site of Hepatitis B virus genomic RNA to asses their efficiency in transcription.

NMR Structural Studies on a DNA Four-Way Junction: Stacking Preference and Localization of the Metal-ion Binding Site.

Abstract The stacking preference of a DNA Four-Way junction (4H), with a novel junction sequence, has been determined in the presence of magnesium ions as well as in the presence of cobalt(III)hexammine ions by means of NMR spectroscopy. In both cases this 4H has a strong preference (>80%) to fold in an A/D-stacked conformer. NOESY spectra showed intermolecular NOE contacts between 4H protons and the ammine protons of the cobalt(III)hexammine complex. These contacts define the metal-ion binding site, located in the vicinity of the junction. The position is similar to the observed site in a recent X-ray structure of a RNA/DNA hybrid 4H and consistent with the position deduced from an uranyl ion photoprobing study on 4Hs with different sequences.

Backbone dynamics of azurin in solution: slow conformational change associated with deprotonation of histidine 35.

15N relaxation measurements have been performed on the type Iota blue copper protein azurin from Pseudomonas aeruginosa. The relaxation times show that one loop (residues 103-108) and one turn (residues 74-77) display fast internal motions. The rest of the protein is rigid with an average order parameter S(2) of 0.85 +/- 0. 05. The copper binding site shows the same degree of rigidity even though is it composed of several loops and lies outside the beta-sheet sandwich. Substantial exchange broadening was found for a number of residues surrounding the side chain of His-35. The average exchange rate has been determined from NMR exchange spectroscopy experiments and is 45 +/- 6 s(-)(1) at 41 degrees C. The exchange broadening is caused by the protonation/deprotonation equilibrium of His-35. The NMR results indicate that the two structures of azurin observed by X-ray diffraction of crystals at pH 5.5 and 9.0 [Nar, H., Messerschmidt, A., Huber, R., Van de Kamp, M., Canters, G. W. (1991) J. Mol. Biol. 221, 765-772] are present in solution and that they interconvert slowly.

Structure and mechanism of formation of the H-y5 isomer of an intramolecular DNA triple helix.

H-DNA, thought to play a regulatory role in transcription, exists in two isomeric forms, H-y3 and H-y5. We present the first solution structure of a DNA fragment representing the H-y5 fold. The structure shows the H-y5 triple helix, and for the first time how in an H-DNA isomer the purine strand extension interacts with the triplex loop. It gives direct insight into the mechanism of H-DNA formation, and explains a host of biochemical and biophysical data on the relative stability of the H-DNA isomers. In addition, the observed interaction of the purine strand extension and the triplex loop provides new clues to the design of clamp-type triple helix-forming oligonucleotides.

A high-resolution HCANH experiment with enhanced sensitivity via multiple quantum line narrowing.

We report a 3D constant-time HCANH experiment (CTSL-HCANH) that uses the slower relaxation of multiple-quantum coherence to increase sensitivity and provides high C(α) resolution. In this experiment the H(α) of the (H(α), C(α)) multiple quanta are selectively spin locked, so that H(α) chemical shift evolution and (1) H-(1)H J-dephasing become ineffective during the relatively long delay needed for C(α) to N coherence transfer. As compared to an HCANH experiment that uses C(α) single-quantum coherence, an average enhancement of 20% was observed on calmodulin in complex with the binding domain of the transcription factor SEF2-1. Compared to CBCANH the signal intensity is approximately twice as good. The favorable relaxation properties of multiple quanta, together with the outstanding C(α) resolution, make the experiment a very good complement to CBCANH and CBCA(CO)NH for sequential assignment of larger proteins for which deuteration is not yet necessary.

Structure of the 3'-hairpin of the TYMV pseudoknot: preformation in RNA folding.

The solution structure of an RNA-hairpin present in the pseudoknot, which is found at the 3'-terminus of turnip yellow mosaic virus genomic RNA, has been solved by nuclear magnetic resonance spectroscopy. The loop, which contains the sequence 5'-GGGUCA-3', was found to be highly structured and, contrary to expectations, does not attain its stability through GA or GC base pair formation but by triple interactions between the tilted adenosine and the minor groove sides of the first two guanosines. Interestingly, a very similar conformation was found for the cognate pseudoknot, implying that the 3'-hairpin is preformed for folding into a pseudoknotted structure. These findings suggest a mechanism of 'predetermined-fit' as a principle in RNA folding.

NMR structure of a classical pseudoknot: interplay of single- and double-stranded RNA.

Pseudoknot formation folds the 3' ends of many plant viral genomic RNAs into structures that resemble transfer RNA in global folding and in their reactivity to transfer RNA-specific proteins. The solution structure of the pseudoknotted T arm and acceptor arm of the transfer RNA-like structure of turnip yellow mosaic virus (TYMV) was determined by nuclear magnetic resonance (NMR) spectroscopy. The molecule is stabilized by the hairpin formed by the 5' end of the RNA, and by the intricate interactions related to the loops of the pseudoknot. Loop 1 spans the major groove of the helix with only two of its four nucleotides. Loop 2, which crosses the minor groove, interacts closely with its opposing helix, in particular through hydrogen bonds with a highly conserved adenine. The structure resulting from this interaction between the minor groove and single-stranded RNA at helical junctions displays internal mobility, which may be a general feature of RNA pseudoknots that regulates their interaction with proteins or other RNA molecules.

The detailed structure of tandem G.A mismatched base-pair motifs in RNA duplexes is context dependent.

The solution structure of the RNA duplex (rGGGCUGAAGCCCU), containing tandem G.A mismatches has been determined by NMR spectroscopy and restrained molecular dynamics. A homonuclear 3D TOCSY-NOESY was used to derive 18 to 30 distance restraints per nucleotide, as well as all gamma torsion angles and sugar puckers for the central UGAA part of the molecule. Using these constraints, together with cross-strand distances, involving exchangeable imino protons, and essentially all other torsion angles that can accurately be determined (i.e. beta, epsilon) otherwise, the structure of the UGAA domain could be determined with high precision (r.m.s.d. 0.62 A), without the aid of isotopically enriched RNA. The G.A base-pairs are of the sheared pairing type, with both nucleotides in the anti conformation, and hydrogen bonds between the guanine 2-amino and the adenine N7 and between the guanine N3 and the adenine 6-amino. Surprisingly the sugar of the guanosine of the G.A. mismatch adopts a 2'-endo sugar pucker conformation. Comparison with other RNA structures, in which two such G.A base-pairs are formed reveals that this detailed structure depends on the identity of the base 5' to the guanosine in the tandem G.A base-pairs. A geometrical model for the incorporation of sheared tandem G.A base-pairs in A-form helices is formulated, which explains the distinct different stacking properties and helical parameters in sequences containing tandem, sheared G.A base-pairs.

Structural features of the DNA hairpin d(ATCCTA-GTTA-TAGGAT): formation of a G-A base pair in the loop.

The three-dimensional structure of the hairpin formed by d(ATCCTA-GTTA-TAGGAT) has been determined by means of two-dimensional NMR studies, distance geometry and molecular dynamics calculations. The first and the last residues of the tetraloop of this hairpin form a sheared G-A base pair on top of the six Watson-Crick base pairs in the stem. The glycosidic torsion angles of the guanine and adenine residues in the G-A base pair reside in the anti and high- anti domain ( approximately -60 degrees ) respectively. Several dihedral angles in the loop adopt non-standard values to accommodate this base pair. The first and second residue in the loop are stacked in a more or less normal helical fashion; the fourth loop residue also stacks upon the stem, while the third residue is directed away from the loop region. The loop structure can be classified as a so-called type-I loop, in which the bases at the 5'-end of the loop stack in a continuous fashion. In this situation, loop stability is unlikely to depend heavily on the nature of the unpaired bases in the loop. Moreover, the present study indicates that the influence of the polarity of a closing A.T pair is much less significant than that of a closing C.G base pair.

Analysis of (1)H chemical shifts in DNA: Assessment of the reliability of (1)H chemical shift calculations for use in structure refinement.

The reliability of (1)H chemical shift calculations for DNA is assessed by comparing the experimentally and calculated chemical shifts of a reasonably large number of independently determined DNA structures. The calculated chemical shifts are based on semiempirical relations derived by Giessner-Prettre and Pullman [(1987) Q. Rev. Biophys., 20, 113-172]. The standard deviation between calculated and observed chemical shifts is found to be quite small, i.e. 0.17 ppm. This high accuracy, which is achieved without parameter adjustment, makes it possible to analyze the structural dependencies of chemical shifts in a reliable fashion. The conformation-dependent (1)H chemical shift is mainly determined by the ring current effect and the local magnetic anisotropy, while the third possible effect, that of the electric field, is surprisingly small. It was further found that for a double helical environment, the chemical shift of the sugar protons, H2' to H5'', is mainly affected by the ring current and magnetic anisotropy of their own base. Consequently, the chemical shift of these sugar protons is determined by two factors, namely the type of base to which the sugar ring is attached, C, T, A, or G, and secondly by the χ-angle. In particular, the H2' shift varies strongly with the χ-angle, and strong upfield H2' shifts directly indicate that the χ-angle is in the syn domain. The H1' shift is not only strongly affected by its own base, but also by its 3'-neighboring base. On the other hand, base protons, in particular H5 of cytosine and methyl protons of thymine, are affected mainly by the 5'-neighboring bases, although some effect (0.2 ppm) stems from the 3'-neighboring base. The H2 protons are mainly affected by the 3'-neighboring base. As a result of these findings a simple scheme is proposed for sequential assignment of resonances from B-helices based on chemical shifts.

The transition from a neutral-pH double helix to a low-pH triple helix induces a conformational switch in the CCCG tetraloop closing a Watson-Crick stem.

The CCCG-loop in a DNA fragment, which is capable of forming an intramolecular triple helix as well as a hairpin structure, was investigated by NMR and molecular modeling studies. The structure of this loop is found as a type II conformation, one of the three commonly observed folding patterns of tetraloops, irrespective of the geometry of the underlying helix. In each situation, the loop exhibits a base-pair between the first cytosine and the guanine residue of the loop. The geometry of this base-pair, however, depends upon the circumstances. At neutral pH, in the hairpin form of the molecule, a Watson-Crick C.G base-pair is formed, whereas at low pH, when the strand exists as an intramolecular triple helix, a Hoogsteen C(+)-G base-pair is present. We used molecular modeling to lay the foundations for understanding the observed conformational switch. A lower amount of strain, related to the short C1'-C1' of the base-pair, and protonation effects of the structure comprising the Hoogsteen base-pair turn out to outweigh the effects of a more stable base-pair, improved stacking and more favorable interactions in the minor groove of the structure comprising the Watson-Crick C.G base-pair. The models also provide an explanation for the general preference of loops meeting the consensus sequence--d(CYNG)--to fold into a type II conformation, i.e. with the base of second loop residue turned into the minor groove.

Through-bond correlation of adenine H2 and H8 protons in unlabeled DNA fragments by HMBC spectroscopy.

A new application of the HMBC experiment is presented that provides a useful means to discriminate between H2 and H8 proton resonances, to assign the base proton resonances to the various residue types and, most importantly, to correlate the H2 and H8 protons for adenine or inosine residues in natural abundance 13C fragments. The utility of this experiment is demonstrated for an unlabeled DNA 20-mer. Thanks to the obtained results, preliminary conclusions could be drawn regarding the molecular conformations of the non-canonical G/I-A base pairs in the hairpin formed by this fragment.