Metabolomic profiling in multiple sclerosis: insights into biomarkers and pathogenesis.

Metabolomics enables the provision of sensitive bio-markers of disease. We performed 800 MHz (1)H-nuclear magnetic resonance (NMR) spectroscopic analyses of cerebrospinal fluid (CSF) specimens to identify biomarkers of multiple sclerosis (MS), yielding reproducible detection of 15 metabolites from MS (n=15) and non-MS (n=17) patients. Mean levels of choline, myo-inositol and threonate were increased, whereas 3-hydroxybutyrate, citrate, phenylalanine, 2-hydroxyisovalerate and mannose were decreased in MS-derived CSF (p<0.05), suggesting alterations to energy and phospholipid metabolism. Multivariate hierarchal cluster analysis indicated a high correlation within the metabolite profiles, significantly clustering samples into the two clinical groups, which was corroborated using principal components analysis. CSF metabolomics have the capacity to yield quantitative biomarkers and insights into the pathogenesis of MS.

Caenorhabditis elegans diet significantly affects metabolic profile, mitochondrial DNA levels, lifespan and brood size.

Diet can have profound effects on an organism's health. Metabolic studies offer an effective way to measure and understand the physiological effects of diet or disease. The metabolome is very sensitive to dietary, lifestyle and genetic changes. Caenorhabditis elegans, a soil nematode, is an attractive model organism for metabolic studies because of the ease with which genetic and environmental factors can be controlled. In this work, we report significant effects of diet, mutation and RNA interference on the C.elegans metabolome. Two strains of Escherichia coli, OP50 and HT115 are commonly employed as food sources for maintaining and culturing the nematode. We studied the metabolic and phenotypic effects of culturing wild-type and mutant worms on these two strains of E. coli. We report significant effects of diet on metabolic profile, on mitochondrial DNA copy number and on phenotype. The dietary effects we report are similar in magnitude to the effects of mutations or RNA interference-mediated gene suppression. This is the first critical evaluation of the physiological and metabolic effects on C.elegans of two commonly used culture conditions.

Calcium-induced peptide association to form an intact protein domain: 1H NMR structural evidence.

The 70-residue carboxyl-terminal domain of the muscle contractile protein troponin-C contains two helix-loop-helix calcium (Ca)-binding sites that are related to each other by approximate twofold rotational symmetry. Hydrophobic residues from the helices and a short three residue beta sheet at the interface of the two sites act to stabilize the protein domain in the presence of Ca. A synthetic 34-residue peptide representing one of these sites (site III) has been synthesized and studied by H-1 nuclear magnetic resonance (NMR) spectroscopy. In solution this peptide undergoes a Ca-induced conformational change to form the helix-loop-helix Ca-binding motif. Two-dimensional nuclear Overhauser effect spectra have provided evidence for the formation of a beta sheet and interactions between several hydrophobic residues from opposing helices as found in troponin-C. It is proposed that a symmetric two-site dimer similar in tertiary structure to the carboxyl-terminal domain of troponin-C forms from the assembly of two site III peptides in the Ca-bound form.

The nonhelical structure of antifreeze protein type III.

Antifreeze proteins (AFPs) are present in the blood of some marine fishes and inhibit the growth of ice crystals at subzero temperatures by adsorption to the ice lattice. The solution structure of a Type III AFP was determined by two-dimensional nuclear magnetic resonance spectroscopy. These measurements indicate that this 66-residue protein has an unusual fold in which eight beta strands form two sheets of three antiparallel strands and one sheet of two antiparallel strands, and the triple-stranded sheets are packed orthogonally into a beta sandwich. This structure is completely different from the amphipathic, helical structure observed for Type I AFPs.

Neutrophil activation by monomeric interleukin-8.

Interleukin-8 (IL-8), a pro-inflammatory protein, has been shown by nuclear magnetic resonance (NMR) and x-ray techniques to exist as a homodimer. An IL-8 analog was chemically synthesized, with the amide nitrogen of leucine-25 methylated to selectivity block formation of hydrogen bonds between monomers and thereby prevent dimerization. This analog was shown to be a monomer, as assessed by analytical ultracentrifugation and NMR. Nevertheless, it was equivalent to IL-8 in assays of neutrophil activation, which indicates that the monomer is a functional form of IL-8.

Thermodynamic insights into proteins from NMR spin relaxation studies.

NMR spin relaxation measurements of picosecond to nanosecond timescale backbone and sidechain fluctuations of protein molecules, and subsequent entropic interpretation yield interesting, but sometimes counterintuitive, insights into proteins. The stabilities of proteins and protein interactions are achieved through enthalpy-entropy compensation, which is partitioned between the backbone and sidechains depending on the nature of the system.

Antifreeze proteins.

Antifreeze proteins comprise a structurally diverse class of proteins that inhibit the growth of ice. Recently, new AFP types have been discovered; more active AFPs have been isolated; antecedents have been recognized supporting the notion of recent, multiple origins; and detailed structures have emerged leading to models for their adsorption to ice.

Refined solution structure of type III antifreeze protein: hydrophobic groups may be involved in the energetics of the protein-ice interaction.

BACKGROUND: Antifreeze proteins are found in certain fish inhabiting polar sea water. These proteins depress the freezing points of blood and body fluids below that of the surrounding sea water by binding to and inhibiting the growth of seed ice crystals. The proteins are believed to bind irreversibly to growing ice crystals in such a way as to change the curvature of the ice-water interface, leading to freezing point depression, but the mechanism of high-affinity ice binding is not yet fully understood. RESULTS: The solution structure of the type III antifreeze protein was determined by multidimensional NMR spectroscopy. Twenty-two structures converged and display a root mean square difference from the mean of 0.26 A for backbone atoms and 0.62 A for all non-hydrogen atoms. The protein exhibits a compact fold with a relatively large hydrophobic core, several short and irregular beta sheets and one helical turn. The ice-binding site, which encompasses parts of the C-terminal sheet and a loop, is planar and relatively nonpolar. The site is further characterized by the low solvent accessibilities and the specific spatial arrangement of the polar side-chain atoms of the putative ice-binding residues Gln9, Asn14, Thr15, Thr18 and Gln44. CONCLUSIONS: In agreement with the adsorption-inhibition mechanism of action, interatomic distances between active polar protein residues match the spacing of water molecules in the prism planes (¿10&1macr;0¿) of the hexagonal ice crystal. The particular side-chain conformations, however, limit the number and strength of possible proten-ice hydrogen bonds. This suggests that other entropic and enthalpic contributions, such as those arising from hydrophobic groups, could play a role in the high-affinity protein-ice adsorption.

Improved synthetic methods for the selective deuteration of aromatic amino acids: applications of selective protonation towards the identification of protein folding intermediates through nuclear magnetic resonance.

In this report we describe several novel methods for the preparation of selectively deuterated aromatic amino acids. New syntheses for [2,3,5,6-2H4]phenylalanine and [2,4,6,7-2H4]tryptophan, as well as improved catalytic exchange methods for [2,3,5,6-2H4]tyrosine and [2,3,4,5,6-2H5]phenylalanine are presented. Isotopic substitution levels for all compounds are generally found to be greater than 95%. Biosynthetic incorporation of these amino acids is also shown to be possible with little or no evidence of isotopic scrambling. The products from these new syntheses, in combination with other selectively deuterated aromatic amino acids, are found to permit group-specific 'single-proton' labelling of proteins. This highly-efficient and very cost-effective method of selective protonation is shown to produce greatly simplified 1H-NMR spectra of the aromatic region of proteins. The utility of this approach to isotopic editing is demonstrated with the identification of a transient folding intermediate of Escherichia coli thioredoxin which is undetectable by standard 2-D NMR techniques.

Interaction of troponin I and troponin C: use of the two-dimensional transferred nuclear Overhauser effect to determine the structure of a Gly-110 inhibitory troponin I peptide analog when bound to cardiac troponin C.

The structure of a peptide analog of the inhibitory region of cardiac troponin-I (N-acetyl-G110-TnI(104-115) amide) when bound to cardiac troponin-C has been determined by 2-dimensional 1H-NMR techniques. The bound structure determined for this peptide is similar to that determined previously for the skeletal peptide (which has a proline at position 110) bound to skeletal troponin-C (Campbell and Sykes (1991) J. Mol. Biol. 222, 405-421). This structure shows a helical like peptide backbone 'bent' around P109-G110 to bring the hydrophobic residues F106, L111 and V114 closer together. The other 'side' of this structure is surrounded by the basic residues extending outwards towards the protein or solution. While the bound structures of the cardiac and skeletal peptides are shown to be quite similar, the cardiac peptide appears more flexible near the central glycine residue.

Recombinant locust apolipophorin III: characterization and NMR spectroscopy.

Apolipophorin III (apoLp-III) from the locust Locusta migratoria is an exchangeable apolipoprotein that reversibly binds to lipoproteins. During lipid binding the protein has been proposed to undergo a major conformational change. To study the mechanism of lipid binding we have cloned and expressed recombinant protein in bacteria, permitting stable isotope enrichment for heteronuclear NMR spectroscopy and site-directed mutagenesis. The cDNA coding for apoLp-III was subcloned into the pET expression vector and transformed into Escherichia coli cells. Induction of expression resulted in the specific appearance of apoLp-III in the cell culture medium, indicating it escaped the bacteria without lysis. The protein was purified from the cell-free supernatant by reversed-phase HPLC, characterized and compared to the natural protein isolated from locust hemolymph. SDS-PAGE revealed the recombinant protein has a molecular mass of approximately 17 kDa, similar to that of deglycosylated natural apoLp-III. Monoclonal antibodies were used to detect recombinant apoLp-III in the cells as well as in cell-free medium of induced bacterial cultures. Amino acid sequencing and analysis confirmed the identity of the recombinant protein as L. migratoria apoLp-III. Circular dichroism spectroscopy of recombinant and natural apoLp-III showed similar spectra, both displaying high contents of alpha-helical secondary structure. Denaturation studies of lipid-free apoLp-III with guanidine hydrochloride showed that both proteins have similar denaturation midpoints and DeltaG values indicating similar protein stability. The natural and recombinant protein were functional in lipoprotein binding assays. Using recombinant protein, uniformly and specifically labeled with 15N-amino acids, two dimensional 1H-15N heteronuclear single quantum correlation spectra were obtained. The spectra revealed excellent chemical shift dispersion in both the 1H and 15N dimensions with a well defined resonance pattern. Studies with 15N-leucine specifically labeled apoLp-III in the presence and absence of the micelle forming lipid, dodecylphosphocholine, provided evidence for a significant conformational change upon lipid association.

Detergent-solubilized M13 coat protein exists as an asymmetric dimer. Observation of individual monomers by 15N, 13C and 1H nuclear magnetic resonance spectroscopy.

M13 coat protein is a simple integral membrane protein isolated from the filamentous coliphage M13. Isotopic labels (13C and 15N) may be incorporated biosynthetically into the protein backbone. 13C nuclear magnetic resonance spectroscopy of carbonyl carbon atoms and two-dimensional 1H-detected 15N-1H heteronuclear shift correlation of coat protein in dodecylsulphate micelles have shown many residues throughout the protein to give rise to two distinct resonances of equal intensity. Chemical shift differences between the two forms are small, indicating the existence of two slightly different but equally populated conformational states. We suggest that the two conformers correspond to the inequivalent monomers of an asymmetric coat protein dimer and propose a mechanism for the generation of such a dimer.

Interaction of troponin I and troponin C. Use of the two-dimensional nuclear magnetic resonance transferred nuclear Overhauser effect to determine the structure of the inhibitory troponin I peptide when bound to skeletal troponin C.

We have used two-dimensional 1H nuclear magnetic resonance spectroscopy to determine the structure of the synthetic inhibitory peptide N alpha-acetyl TnI(104-115) amide bound to calcium-saturated skeletal troponin C (TnC). Conformational changes in the peptide induced by the formation of the troponin I (TnI) peptide-TnC complex were followed by the study of the transferred nuclear Overhauser effect, a technique that allows one to determine the structure of a ligand bound to a macromolecule. The structure of the bound TnI peptide reveals an amphiphilic alpha-helix, distorted around the two central proline residues. The central bend in the peptide functions to bring the residues on the hydrophobic face into closer proximity with each other, thereby forming a small hydrophobic pocket. The hydrophilic, basic residues extend off the opposite face of the peptide. Hydrophobic surfaces on TnC that become exposed upon binding of calcium are involved in the binding of the TnI peptide, but electrostatic interactions also contribute to the strength of the interaction. The role of amphiphilic helices in the targeting of calcium-binding proteins such as troponin C will be discussed.

Relationship between nuclear magnetic resonance chemical shift and protein secondary structure.

An analysis of the 1H nuclear magnetic resonance chemical shift assignments and secondary structure designations for over 70 proteins has revealed some very strong and unexpected relationships. Similar studies, performed on smaller databases, for 13C and 15N chemical shifts reveal equally strong correlations to protein secondary structure. Among the more interesting results to emerge from this work is the finding that all 20 naturally occurring amino acids experience a mean alpha-1H upfield shift of 0.39 parts per million (from the random coil value) when placed in a helical configuration. In a like manner, the alpha-1H chemical shift is found to move downfield by an average of 0.37 parts per million when the residue is placed in a beta-strand or extended configuration. Similar changes are also found for amide 1H, carbonyl 13C, alpha-13C and amide 15N chemical shifts. Other relationships between chemical shift and protein conformation are also uncovered; in particular, a correlation between helix dipole effects and amide proton chemical shifts as well as a relationship between alpha-proton chemical shifts and main-chain flexibility. Additionally, useful relationships between alpha-proton chemical shifts and backbone dihedral angles as well as correlations between amide proton chemical shifts and hydrogen bond effects are demonstrated.

Solution conformation of purine-pyrimidine DNA octamers using nuclear magnetic resonance, restrained molecular dynamics and NOE-based refinement.

The solution structures of two alternating purine-pyrimidine octamers, [d(G-T-A-C-G-T-A-C)]2 and the reverse sequence [d(C-A-T-G-C-A-T-G)]2, are investigated by using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Chemical shift assignments are obtained for non-exchangeable protons by a combination of two-dimensional correlation and nuclear Overhauser enhancement (NOE) spectroscopy experiments. Distances between protons are estimated by extrapolating distances derived from time-dependent NOE measurements to zero mixing time. Approximate dihedral angles are determined within the deoxyribose ring from coupling constants observed in one and two-dimensional spectra. Sets of distance and dihedral determinations for each of the duplexes form the bases for structure determination. Molecular dynamics is then used to generate structures that satisfy the experimental restraints incorporated as effective potentials into the total energy. Separate runs start from classical A and B-form DNA and converge to essentially identical structures. To circumvent the problems of spin diffusion and differential motion associated with distance measurements within molecules, models are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated using a full matrix analysis procedure. The refined structures generally have the global features of B-type DNA. Some, but not all, variations in dihedral angles and in the spatial relationships of adjacent base-pairs are observed to be in synchrony with the alternating purine-pyrimidine sequence.

Insights into the mechanism of heterodimerization from the 1H-NMR solution structure of the c-Myc-Max heterodimeric leucine zipper.

The oncoprotein c-Myc (a member of the helix-loop-helix-leucine zipper (b-HLH-LZ) family of transcription factors) must heterodimerize with the b-HLH-LZ Max protein to bind DNA and activate transcription. It has been shown that the LZ domains of the c-Myc and Max proteins specifically form a heterodimeric LZ at 20 degreesC and neutral pH. This suggests that the LZ domains of the c-Myc and Max proteins are playing an important role in the heterodimerization of the corresponding gene products in vivo. Initially, to gain an insight into the energetics of heterodimerization, we studied the stability of N-terminal disulfide-linked versions of the c-Myc and Max homodimeric LZs and c-Myc-Max heterodimeric LZ by fitting the temperature-induced denaturation curves monitored by circular dichroism spectroscopy. The c-Myc LZ does not homodimerize (as previously reported) and the c-Myc-Max heterodimeric LZ is more stable than the Max homodimeric LZ at 20 degreesC and pH 7.0. In order to determine the critical interhelical interactions responsible for the molecular recognition between the c-Myc and Max LZs, the solution structure of the disulfide-linked c-Myc-Max heterodimeric LZ was solved by two-dimensional 1H-NMR techniques at 25 degreesC and pH 4.7. Both LZs are alpha-helical and the tertiary structure depicts the typical left-handed super-helical twist of a two-stranded parallel alpha-helical coiled-coil. A buried salt bridge involving a histidine on the Max LZ and two glutamate residues on the c-Myc LZ is observed at the interface of the heterodimeric LZ. A buried H-bond between an asparagine side-chain and a backbone carbonyl is also observed. Moreover, evidence for e-g interhelical salt bridges is reported. These specific interactions give insights into the preferential heterodimerization process of the two LZs. The low stabilities of the Max homodimeric LZ and the c-Myc-Max heterodimeric LZ as well as the specific interactions observed are discussed with regard to regulation of transcription in this family of transcription factors.

Backbone and methyl dynamics of the regulatory domain of troponin C: anisotropic rotational diffusion and contribution of conformational entropy to calcium affinity.

The N-terminal domain (residues 1 to 90) of chicken skeletal troponin C (NTnC) regulates muscle contraction upon the binding of a calcium ion to each of its two calcium binding loops. In order to characterize the backbone dynamics of NTnC in the apo state (NTnC-apo), we measured and carefully analyzed 15N NMR relaxation parameters T1, T2 and NOE at 1H NMR frequencies of 500 and 600 MHz. The overall rotational correlation time of NTnC-apo at 29.6 degrees C is 4.86 (+/-0.15) ns. The experimental data indicate that the rotational diffusion of NTnC-apo is anisotropic with a diffusion anisotropy, D parallel/D perpendicular, of 1.10. Additionally, the dynamic properties of side-chains having a methyl group were derived from 2H relaxation data of CH2D groups of a partially deuterated sample. Based on the dynamic characteristics of TnC, two different levels of "fine tuning" of the calcium affinity are presented. Significantly lower backbone order parameters (S2), were observed for calcium binding site I relative to site II and the contribution of the bond vector fluctuations to the conformational entropy of sites I and II was calculated. The conformational entropy loss due to calcium binding (DeltaDeltaSp) differs by 1 kcal/mol between sites I and II. This is consistent with the different dissociation constants previously measured for sites I and II of 16 microM and 1. 7 microM, respectively. In addition to the direct role of binding loop dynamics, the side-chain methyl group dynamics play an indirect role through the energetics of the calcium-induced structural change from a closed to an open state. Our results show that the side-chains which will be exposed upon calcium binding have reduced motion in the apo state, suggesting that conformational entropic contributions can be used to offset the free energy cost of exposing hydrophobic groups. It is clear from this work that a complete determination of their dynamic characteristics is necessary in order to fully understand how TnC and other proteins are fine tuned to appropriately carry out their function.

Interaction of the receptor binding domains of Pseudomonas aeruginosa pili strains PAK, PAO, KB7 and P1 to a cross-reactive antibody and receptor analog: implications for synthetic vaccine design.

The four synthetic peptide antigens, PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, correspond in amino acid sequence to the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin. The NMR solution structures of the trans forms of the peptides show conserved beta-turns which have been implicated in antibody and receptor recognition. The interactions between these peptides and a cross-reactive monoclonal antibody, PAK-13, have been studied using two-dimensional (1)H NMR spectroscopy in order to map the antigenic determinants recognized by the antibody. Residues for which spectral changes were observed upon antibody binding differed from peptide to peptide but were mostly confined to one or both of the turn regions and to the hydrophobic pockets. Conformational changes in the beta-turns and hydrophobic pockets of these peptides upon antibody binding were also monitored by examination of the pattern of nuclear Overhauser effects (NOEs) versus transferred nuclear Overhauser effects (TRNOEs) for the free versus the bound peptides. Although TRNOEs developed strongly between side chain resonances in the hydrophobic pockets of the peptides, no additional backbone TRNOEs were observed in the presence of antibody, suggesting no major conformational changes in the secondary structures of the peptides upon binding. This implies a flexible antibody combining site, a feature which is discussed with respect to cross-reactivity, strain specificity, and the design of a synthetic peptide vaccine effective against a broad spectrum of P. aeruginosa strains. The binding of the PAK peptide to a disaccharide receptor analog, (beta GalNAc(1-4)beta Gal), was also studied using (1)H NMR in order to map the "adhesintope" recognized by the receptor. Spectral changes observed in the peptide spectrum with the binding of receptor were similar to those seen for the binding of antibody, suggesting that the epitope recognized by the antibody is structurally coincident with the adhesintope recognized by the receptor. The relevancy of this result is discussed with respect to immunogenicity versus pathogenicity, and the proper design of a vaccine which could prevent the mutational escape of the pathogen away from the host's defence systems.

Recommendations for the presentation of NMR structures of proteins and nucleic acids.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Determination of the conformations of bound peptides using NMR-transferred nOe techniques.

NMR-transferred nOe spectroscopy provides a means for determining the structure of a peptide bound to a macromolecular target when the complex is too large to be studied by conventional NMR techniques. Recent examples of structures that have been determined by NMR-transferred nOe techniques include platelet-receptor peptide bound to thrombin, cholera toxin peptide bound to antibodies, troponin I peptide bound to troponin C and the vsv-C peptide bound to chaperones DnaK and GroEL.