Assessment of cognition in the elderly: pieces of a complex puzzle

A suite of programs called CAMRA (Computer Aided Magnetic Resonance Assignment) has been developed for computer assisted residue-specific assignments of proteins. CAMRA consists of three units: ORB, CAPTURE and PROCESS. ORB predicts NMR chemical shifts for unassigned proteins using a chemical shift database of previously assigned homologous proteins supplemented by a statistically derived chemical shift database in which the shifts are categorized according to their residue, atom and secondary structure type. CAPTURE generates a list of valid peaks from NMR spectra by filtering out noise peaks and other artifacts and then separating the derived peak list into distinct spin systems. PROCESS combines the chemical shift predictions from ORB with the spin systems identified by CAPTURE to obtain residue specific assignments. PROCESS ranks the top choices for an assignment along with scores and confidence values. In contrast to other auto-assignment programs, CAMRA does not use any connectivity information but instead is based solely on matching predicted shifts with observed spin systems. As such, CAMRA represents a new and unique approach for the assignment of protein NMR spectra. CAMRA will be particularly useful in conjunction with other assignment methods and under special circumstances, such as the assignment of flexible regions in proteins where sufficient NOE information is generally not available. CAMRA was tested on two medium-sized proteins belonging to the chemokine family. It was found to be effective in predicting the assignment providing a database of previously assigned proteins with at least 30% sequence identity is available. CAMRA is versatile and can be used to include and evaluate heteronuclear and three-dimensional experiments.     

Solution structure of human intestinal fatty acid binding protein: implications for ligand entry and exit

The human intestinal fatty acid binding protein (I-FABP) is a small (131 amino acids) protein which binds dietary long-chain fatty acids in the cytosol of enterocytes. Recently, an alanine to threonine substitution at position 54 in I-FABP has been identified which affects fatty acid binding and transport, and is associated with the development of insulin resistance in several populations including Mexican-Americans and Pima Indians. To investigate the molecular basis of the binding properties of I-FABP, the 3D solution structure of the more common form of human I-FABP (Ala54) was studied by multidimensional NMR spectroscopy. Recombinant I-FABP was expressed from E. coli in the presence and absence of 15N-enriched media. The sequential assignments for non-delipidated I-FABP were completed by using 2D homonuclear spectra (COSY, TOCSY and NOESY) and 3D heteronuclear spectra (NOESY-HMQC and TOCSY-HMQC). The tertiary structure of human I-FABP was calculated by using the distance geometry program DIANA based on 2519 distance constraints obtained from the NMR data. Subsequent energy minimization was carried out by using the program SYBYL in the presence of distance constraints. The conformation of human I-FABP consists of 10 antiparallel beta-strands which form two nearly orthogonal beta-sheets of five strands each, and two short alpha-helices that connect the beta-strands A and B. The interior of the protein consists of a water-filled cavity between the two beta-sheets. The NMR solution structure of human I-FABP is similar to the crystal structure of rat I-FABP. The NMR results show significant conformational variability of certain backbone segments around the postulated portal region for the entry and exit of fatty acid ligand.     

Pterulinic acid and pterulone, two novel inhibitors of NADH:ubiquinone oxidoreductase (complex I) produced by a Pterula species. II. Physico-chemical properties and structure elucidation

The structures of two novel fungal antibiotics, isolated from a Pterula species, that interfere with the NADH:ubiquinone oxidoreductase and inhibit the respiration of eucaryotes, were determined by spectroscopic techniques. Both compounds, pterulinic acid (1a) and pterulone (2), contain a 1-benzoxepin ring system and are chlorinated. Pterulinic acid (1a), which was obtained as a 1:5 inseparable mixture of the two isomers (Z)-1a and (E)-1a, in addition contains a furan. Their structures were determined by mass spectrometry and NMR spectroscopy, and 2D heteronuclear correlation experiments permitted the assignment of all NMR signals.     

13C nuclear magnetic resonance study of 17 alpha-substituted estradiols

We report the 13C NMR data for 20 compounds bearing a substituent (alkyl, alkenyl, alkynyl, alkylamide, spiro-gamma-lactone, phenyl, benzyl, naphthyl, etc.) at the 17 alpha-position of estradiol. The carbon assignments were done using 1D and 2D NMR experiments (distortionless enhancement by polarization transfer, homonuclear correlated spectroscopy, heteronuclear shift correlation, and heteronuclear shift correlation via long-range couplings). Only the chemical shifts of carbons 12-18, which surround the substitution site, were affected by the addition of a substituent. The magnitude of the effects (shielding or deshielding) was influenced by the 17 alpha-substituent. The individual effects at these carbons were sufficiently distinctive to identify specific centers and should be valuable for signal assignment of a variety of 17 alpha-derivatives of estradiol. In addition to carbon-skeleton assignment, we also report the carbon-substituent assignments.     

Characterization of a site-directed mutant of cytochrome b5 designed to alter axial imidazole ligand plane orientation

Mutants of cytochrome b5 were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V mutation achieved this goal through the substitution of a bulkier, hydrophobic ligand for a residue, in the sterically hindered hydrophobic heme binding pocket. Solution structures of mutant and wild-type proteins in the region of the mutation were calculated using restraints obtained from 1H and 15N 2D homonuclear and heteronuclear NMR spectra and 1H-15N 3D heteronuclear NMR spectra. More than 10 restraints per residue were used in the refinement of both structures. Average local rmsd for 20 refined structures was 0.30 A for the wild-type structure and 0.38 A for the A67V mutant. The transfer of amide proton resonance assignments from wild-type to the mutant protein was achieved through overlays of 15N-1H heteronuclear correlation spectra of the reduced proteins. Side chain assignments and sequential assignments were established using conventional assignment strategies. Calculation of the orientation of the components of the anisotropic paramagnetic susceptibility tensor, using methods similar to procedures applied to the wild-type protein, shows that the orientation of the in-plane components are identical in the wild-type and mutant proteins. However, the orientation of the z-component of the susceptibility tensor calculated for the mutant protein differs by 17 degrees for the A-form and by 11 degrees for the B-form from the orientation calculated for the wild-type protein. The rotation of the z-component of the susceptibility tensor (toward the delta meso proton) is in the same direction and is of the same magnitude as the rotation of the H63 imidazole ring induced by mutation.     

Proton nuclear magnetic resonance studies on huwentoxin-I from the venom of the spider Selenocosmia huwena: 1. Sequence-specific 1H-NMR assignments

The complete sequence-specific assignments of resonances in the 1H-NMR spectrum of huwentoxin-I from the Chinese bird spider, Selencocosmia huwena, is described. A combination of two-dimensional NMR experiments including 2D-COSY, 2D-NOESY, and 2D-TOCSY has been employed on samples of the toxin dissolved in D2O and in H2O for assignment purposes. Protons belonging to spin systems for each of the 33 amino acids were identified. The sequence-specific assignments were facilitated by the identification of d alpha N connectivities on the fingerprint regions of the COSY and NOESY spectra and were supported by the identification of dNN and d alpha N connectivities in the TOCSY and NOESY spectra. These studies provide a basis for the determination of the solution-phase conformation of this toxin.     

Characterizing the use of perdeuteration in NMR studies of large proteins: 13C, 15N and 1H assignments of human carbonic anhydrase II

Perdeuteration of all non-exchangeable proton sites can significantly increase the size of proteins and protein complexes for which NMR resonance assignments and structural studies are possible. Backbone 1H, 15N, 13CO, 13C alpha and 13C beta chemical shifts and aliphatic side-chain 13C and 1H(N)/15N chemical shifts for human carbonic anhydrase II (HCA II), a 259 residue 29 kDa metalloenzyme, have been determined using a strategy based on 2D, 3D and 4D heteronuclear NMR experiments, and on perdeuterated 13C/15N-labeled protein. To date, HCA II is one of the largest monomeric proteins studied in detail by high-resolution NMR. Of the backbone resonances, 85% have been assigned using fully protonated 15N and 3C/15N-labeled protein in conjunction with established procedures based on now standard 2D and 3D NMR experiments. HCA II has been perdeuterated both to complete the backbone resonance assignment and to assign the aliphatic side-chain 13C and 1H(N)/15N resonances. The incorporation of 2H into HCA II dramatically decreases the rate of 13C and 1H(N)T2 relaxation. This, in turn, increases the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments. Many otherwise marginal heteronuclear 3D and 4D correlation experiments, which are important to the assignment strategy detailed herein, can now be executed successfully on HCA II. Further analysis suggests that, from the perspective of sensitivity, perdeuteration should allow other proteins with rotational correlation times significantly longer than HCA II (tau c = 11.4 ns) to be studied successfully with these experiments. Two different protocols have been used to characterize the secondary structure of HCA II from backbone chemical-shift data. Secondary structural elements determined in this manner compare favorably with those elements determined from a consensus analysis of the HCA II crystal structure. Finally, having outlined a general strategy for assigning backbone and side-chain resonances in a perdeuterated large protein, we propose a strategy whereby this information can be used to glean more detailed structural information from the partially or fully protonated protein equivalent.     

Solution structure of recombinant human interleukin-6

Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivo and whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five alpha-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 A in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 A for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.     

Cysteinyl-tRNA synthetase from Saccharomyces cerevisiae. Purification, characterization and assignment to the genomic sequence YNL247w

Cysteinyl-tRNA synthetase (CRS) from Saccharomyces cerevisiae was purified 2300-fold with a yield of 33%, to a high specific activity (kcat4.3 s-1 at 25 degrees C for the aminoacylation of yeast tRNACys). SDS-PAGE revealed a single polypeptide corresponding to a molecular mass of 86 kDa. Polyclonal antibodies to the purified protein inactivated CRS activity and detected only one polypeptide of 86 kDa in a yeast extract subjected to SDS-PAGE followed by immunoblotting. In contrast to bacterial CRS which is a monomer of about 50 kDa, the native yeast enzyme behaved as a dimer, as assessed by gel filtration and cross-linking. Its subunit molecular mass is in good agreement with the value of 87.5 kDa calculated for the protein encoded by the yeast genomic sequence YNL247w. The latter was previously tentatively assigned to CRS, based on limited sequence similarities to the corresponding enzyme from other sources. Determination of the amino acid sequence of internal polypeptides derived from the purified yeast enzyme confirmed this assignment. Alignment of the primary sequences of prokaryotic and yeast CRS reveals that the larger size of the latter is accounted for mostly by several insertions within the sequence.     

Partial gene structure and assignment to chromosome 2q37 of the human inwardly rectifying K+ channel (Kir7.1) gene (KCNJ13)

The novel weakly inward rectifying potassium channel Kir7.1 is a low-conductance channel that is predominantly expressed in epithelial cells. Here we describe a partial genomic characterization and the chromosomal assignment of the human Kir7.1 gene (KCNJ13). Analysis of the genomic structure using a PCR-based approach revealed a single 2088-bp intron in the coding region of KCNJ13. PCR analysis of monochromosomal and radiation hybrid panels assigns KCNJ13 to band 2q37 between markers D2S331 and D2S345. In addition, a single nucleotide polymorphism (C524-->T), leading to an exchange of a Thr with an Ile residue at amino acid position 175, was found.     

Classification of amino acid spin systems using PFG HCC(CO)NH-TOCSY with constant-time aliphatic 13C frequency labeling

We have developed a useful strategy for identifying amino acid spin systems and side-chain carbon resonance assignments in small 15N-, 13C-enriched proteins. Multidimensional constant-time pulsed field gradient (PFG) HCC(CO)NH-TOCSY experiments provide side-chain resonance frequency information and establish connectivities between sequential amino acid spin systems. In PFG HCC(CO)NH-TOCSY experiments recorded with a properly tuned constant-time period for frequency labeling of aliphatic 13C resonances, phases of cross peaks provide information that is useful for identifying spin system types. When combined with 13C chemical shift information, these patterns allow identification of the following spin system types: Gly, Ala, Thr, Val, Leu, Ile, Lys, Arg, Pro, long-type (i.e., Gln, Glu and Met), Ser, and AMX-type (i.e., Asp, Asn, Cys, His, Phe, Trp and Tyr).     

Structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c

The following structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c containing D-mannose and D-rhamnose was established using sugar analysis and NMR spectroscopy, including computer-assisted analysis of the 13C NMR spectrum, 2D COSY, H,H-relayed COSY, heteronuclear 13C, 1H correlation (HETCOR), and rotating-frame NOE spectroscopy (ROESY):-->4)-alpha-D-Manp-(1-->3)-beta-D-Rhap-(1-->4) -beta-D-Rhap-(1-->.     

An NMR assignment module implemented in the Gifa NMR processing program

MOTIVATION: Peptide and protein structures are determined daily using NMR spectroscopy. Assignment of the NMR spectra is an important step within the procedure and is usually the limiting one. Computer-aided assignment tools should be user friendly with open architecture to communicate with other programs involved in the structure determination. RESULTS: Here we present an interactive NMR assignment module which provides numerous graphic tools for the user. The module is composed of a database management system-handling peaks, spins and spin-systems. The assignment information is maintained as a set of interrelated associative arrays, which serve as generic high-level data structures. The module is developed in the macro language embedded in the Gifa NMR processing program (Pons et al. , J. Biomol. NMR, 8 , 445-452, 1996). This provides the user with a consistent interface, a set of sophisticated tools, and an easily extendible and customizable environment. AVAILABILITY: The program is available on request from the authors. The Gifa package can be accessed at: ((http://www.cbs. univ-montp1.fr/GIFA)) CONTACT: Marc-Andre.Delsuc@cbs.univ-montp1.fr     

Syntenic assignment of dopamine tautomerase (DCT) to bovine chromosome 12

Heterologous primers were used to amplify an exon and intron-containing segment of the bovine homologue of the human dopachrome tautomerase gene. After confirmation of homology by sequence analysis (exon sequence similarity greater than 90%), bovine-specific primers were developed for synteny mapping purposes. The dopachrome tautomerase gene was assigned to bovine chromosome 12 (BTA12) with 97% concordance to the coagulation factor 10 locus. Together with previous synteny mapping of bovine chromosome 12 genes, fms-related tyrosine kinase, esterase D and 5-hydroxytryptamine receptor 2, this assignment further indicates conservation between human chromosome 13q and bovine chromosome 12.     

1H and 15N nuclear magnetic resonance assignments, secondary structure in solution, and solvent exchange properties of azurin from Alcaligenes denitrificans

Complete sequential 1H and 15N resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) = 14,000, 129 residues) from Alcaligenes denitrificans have been obtained at pH 5.5 and 32 degrees C using homo- and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Comparison of the resonance assignments for the backbone protons with those of Pseudomonas aeruginosa azurin, which is 68% homologous in its amino acid sequence and has a very similar three-dimensional structure, showed a high similarity in chemical shift positions. After adjustment for random coil contributions the mean difference in NH chemical shifts is 0.00 ppm (root mean square width = 0.30 ppm), whereas for C alpha protons the mean difference is 0.09 ppm (root mean square width = 0.23 ppm). Characteristic NOE connectivities and 3JHN alpha values were used to determine the secondary structure of azurin in solution. Two beta-sheets, one helix, and nine tight and four helical turns were identified, and some long-range NOE contacts were found that connect the helix with the beta-sheets. The secondary structure obtained is in agreement with the structure derived from X-ray diffraction data [Baker, E. N. (1988) J. Mol. Biol. 203, 1071-1095]. Studies of the hydration of the protein in the vicinity of the copper ligand residue His117 revealed that the solvent-exposed N epsilon 2 of His117 is in slow exchange with the bulk solvent. However, no evidence was obtained for the presence of a long-lived water molecule at the position corresponding to a well-defined water molecule observed in the crystal structures of A. denitrificans and Ps. aeruginosa azurin.     

Somatic antigens of pseudomonads: structure of the O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247

The O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247, was studied by acid hydrolysis, GLC-MS and 1H and 13C NMR spectroscopy, including 1D and 2D NOE, 2D hybrid TOCSY and ROESY (TORO), and 2D H-detected heteronuclear multiple-bond correlation (HMBC) experiments. The polysaccharide was found to contain L-rhamnose, 3.6-dideoxy-3-[(S)-3-hydroxybutyramido]-D-glucose (D-Qui3NHb), 2-acetamido- 2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido-D-glucose (D-QuiNAc4NHb) and 2-acetamido-2- deoxy-D-galacturonic acid (D-GalNAcA). Partial acid hydrolysis of the polysaccharide resulted in a non-reducing GalNAcA-->QuiNAc4NHb disaccharide with the 3-hydroxybutyryl group glycosylated intramolecularly by the QuiN4N residue. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established:-->4) -alpha-D-GalpNAcA-(1-->3)- alpha-D-QuipNAc4NHb-(1-->2)-beta-D-Quip3NHb-(1-->2)-alpha-L- Rhap(1-->.     

Sequential assignment of 1H, 15N, 13C resonances and secondary structure of human calmodulin-like protein determined by NMR spectroscopy

Human calmodulin-like protein (CLP) is closely related to vertebrate calmodulin, yet its unique cell specific expression pattern, overlapping but divergent biochemical properties, and specific target proteins suggest that it is not an isoform of calmodulin. To gain insight into the structural differences that may underlie the difference target specificities and biochemical properties of CLP when compared to calmodulin, we determined the sequential backbone assignment and associated secondary structure of 144 out of the 148 residues of Ca2+-CLP by using multinuclear multidimensional NMR spectroscopy. Despite a very high overall degree of structural similarity between CLP and calmodulin, a number of significant differences were found mainly in the length of alpha-helices and in the central nonhelical flexible region. Interestingly, the regions of greatest primary sequence divergence between CLP and calmodulin in helices III and VIII displayed only minor secondary structure differences. The data suggest that the distinct differences in target specificity and biochemical properties of CLP and calmodulin result from the sum of several minor structural and side-chain changes spread over multiple domains in these proteins.     

TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins

The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below approximately 25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.     

The A-kinase anchoring domain of type IIalpha cAMP-dependent protein kinase is highly helical

Subcellular localization of the type II cAMP-dependent protein kinase is controlled by interaction of the regulatory subunit with A-Kinase Anchoring Proteins (AKAPs). This contribution examines the solution structure of a 44-residue region that is sufficient for high affinity binding to AKAPs. The N-terminal dimerization domain of the type IIalpha regulatory subunit of cAMP-dependent protein kinase was expressed to high levels on minimal media and uniformly isotopically enriched with 15N and 13C nuclei. Sequence-specific backbone and side chain resonance assignments have been made for greater than 95% of the amino acids in the free dimerization domain using high resolution multidimensional heteronuclear NMR techniques. Contrary to the results from secondary structure prediction algorithms, our analysis indicates that the domain is highly helical with a single 3-5-residue sequence involved in a beta-strand. The assignments and secondary structure analysis provide the basis for analyzing the structure and dynamics of the dimerization domain both free and complexed with specific anchoring proteins.