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At least 235 records · Page 13Linked to original sources
PubMed1996

Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin.

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhancement-derived distance constraints, ten phi torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 angstroms for the backbone and 1.92 angstroms for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well defined, with r.m.s. deviations of 0.64 angstroms for the backbone and 1.51 angstroms for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in an intra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain alpha-helix. This interaction interferes with the inter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin-receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.

Amino Acid Sequence↗
PubMed1998

Structure determination of the three disulfide bond isomers of alpha-conotoxin GI: a model for the role of disulfide bonds in structural stability.

The three possible disulfide bonded isomers of alpha-conotoxin GI have been selectively synthesised and their structures determined by 1H NMR spectroscopy. alpha-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2-7;3-13), GI(2-13;3-7) and GI(2-3;7-13). The NMR structure for the native isomer GI(2-7;3-13) is of excellent quality, with a backbone pairwise RMSD of 0.16 A for a family of 35 structures, and comprises primarily a distorted 310 helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2-7;3-13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in alpha-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2-7;3-13)<GI(2-13;3-7)<GI(2-3;7-13). It was found that the peptide bond joining Cys2 to Cys3 in GI(2-3;7-13) is predominantly trans, rather than cis as theoretically predicted. These structural data are used to interpret the varying nAChR binding of the non-native forms.A model for the binding of native GI(2-7;3-13) to the mammalian nAChR is proposed, with an alpha-subunit binding face made up of Cys2, Asn4, Pro5, Ala6 and Cys7 and a selectivity face, comprised of Arg9 and His10. These two faces orient the molecule between the alpha and delta subunits of the receptor. The structure of the CCNPAC sequence of the native GI(2-7;3-13) is compared to the structure of the identical sequence from the toxic domain of heat-stable enterotoxins, which forms part of the receptor binding region of the enterotoxins, but which has a different disulfide connectivity.

Amino Acid Sequence↗
PubMed1998

Prediction of local structure in proteins using a library of sequence-structure motifs.

We describe a new method for local protein structure prediction based on a library of short sequence pattern that correlate strongly with protein three-dimensional structural elements. The library was generated using an automated method for finding correlations between protein sequence and local structure, and contains most previously described local sequence-structure correlations as well as new relationships, including a diverging type-II beta-turn, a frayed helix, and a proline-terminated helix. The query sequence is scanned for segments 7 to 19 residues in length that strongly match one of the 82 patterns in the library. Matching segments are assigned the three-dimensional structure characteristic of the corresponding sequence pattern, and backbone torsion angles for the entire query sequence are then predicted by piecing together mutually compatible segment predictions. In predictions of local structure in a test set of 55 proteins, about 50% of all residues, and 76% of residues covered by high-confidence predictions, were found in eight-residue segments within 1.4 A of their true structures. The predictions are complementary to traditional secondary structure predictions because they are considerably more specific in turn regions, and may contribute to ab initio tertiary structure prediction and fold recognition.

Algorithms↗
PubMed2000

Generation of deviation parameters for amino acid singlets, doublets and triplets from three-dimentional structures of proteins and its implications for secondary structure prediction from amino acid sequences.

We present a new method, secondary structure prediction by deviation parameter (SSPDP) for predicting the secondary structure of proteins from amino acid sequence. Deviation parameters (DP) for amino acid singlets, doublets and triplets were computed with respect to secondary structural elements of proteins based on the dictionary of secondary structure prediction (DSSP)-generated secondary structure for 408 selected non-homologous proteins. To the amino acid triplets which are not found in the selected dataset, a DP value of zero is assigned with respect to the secondary structural elements of proteins. The total number of parameters generated is 15,432, in the possible parameters of 25,260. Deviation parameter is complete with respect to amino acid singlets, doublets, and partially complete with respect to amino acid triplets. These generated parameters were used to predict secondary structural elements from amino acid sequence. The secondary structure predicted by our method (SSPDP) was compared with that of single sequence (NNPREDICT) and multiple sequence (PHD) methods. The average value of the percentage of prediction accuracy for a helix by SSPDP, NNPREDICT and PHD methods was found to be 57%, 44% and 69% respectively for the proteins in the selected dataset. For b-strand the prediction accuracy is found to be 69%, 21% and 53% respectively by SSPDP, NNPREDICT and PHD methods. This clearly indicates that the secondary structure prediction by our method is as good as PHD method but much better than NNPREDICT method.

Amino Acid Sequence↗
PubMed2002

Three-dimensional solution structure of Tropidechis carinatus venom extract trocarin: a structural homologue of Xa and prothrombin activator.

Trocarin belongs to group D of prothrombin activators derived from snake venom of Tropidechis carinatus and is a rich non-hepatic source of Xa, the only known hepatic prothrombin activator. The structural and functional similarity with Xa makes trocarin an interesting target for exploring the structure-functional relationship with Xa. Herein we report a predicted complete three-dimensional all-atom structural model of trocarin equilibrated in explicit water using 4 ns of molecular dynamics simulation. The tertiary structure was modeled using the structure of human blood coagulation factor Xa. The conformational and structural features of trocarin are then compared with the X-ray crystal and solution simulation structures of human factor Xa. The modeled structure of trocarin has four individual domains (Gla, EGF1, EGF2 and SP) connected along the long axis with similar secondary structural elements to Xa. The simulations suggest that sodium ion binding in the serine protease domain is impaired in trocarin as compared to Xa. In contrast to Xa, for which the sodium ion forms an octahedral coordination network that brings two loop regions connecting four anti-parallel beta-sheets together, we do not find a similar pattern of network in trocarin. We observe that the difference in the binding pattern of sodium ion leads to a approximately 2-A "shrinkage" of the beta2 strand (B2), in comparison to human Xa, as marked by a shorter distance between 189Asp373 (S1-site residue) and 195Ser379 (active-site residue) in the B2 strand. We propose that these differences may be linked to the experimentally observed lower amidolytic activity of trocarin as compared to Xa.

Amino Acid Sequence↗
PubMed1991

Correlation of secondary structure with biological activity for a leader peptide: circular dichroism-derived structure and in vitro biological activities of preproparathyroid hormone peptide and its analogs.

Leader or signal sequences are specialized domains within precursor proteins which serve an essential role in interacting with the cellular secretory apparatus to enable intracellular transport and secretion of proteins. Despite many differences in primary amino acid sequences, signal domains interact with a common set of intracellular components, presumably because the signal sequences share an overall conformational similarity. In a few instances, mutant signal peptides from prokaryotes have been studied and their structures correlated with function (export) in vivo. A series of analogs of the precursor-specific region of preproparathyroid hormone have been prepared which contain substitutions of either proline or a charged amino acid within the hydrophobic core. These synthetic "mutants" have previously been evaluated in several in vitro assays to determine their functionality with regard to protein secretion and suitability as substrates for signal peptidase. The secondary structural content of each peptide, as well as the native sequence and sulfur-free analog, was determined in aqueous and nonaqueous conditions by circular dichroism (CD) as a function of time. The structures obtained were correlated with in vitro bioactivities. Unlike the findings or previous CD studies, all the peptides examined here had low to undetectable alpha-helical content in both aqueous and nonaqueous buffers. The unsubstituted and sulfur-free analogs had high (80-85%) beta-structure in aqueous conditions which was reduced to approximately 30% in nonaqueous solvent. The proline- and charged-substituted peptides contained about half the beta-structure content (35-55%) in aqueous buffer; in nonaqueous solvent their structure was similar to the unsubstituted peptides. The structure-activity correlates found were as follows: a high degree of structure (aqueous conditions) correlated with interaction with signal recognition particle and substrate suitability for signal peptidase; a low degree of structure (nonaqueous environment) correlated with activity in the translocation assay.

Amino Acid Sequence↗
PubMed2004

Structural evidence for guanidine-protein side chain interactions: crystal structure of CutA from Pyrococcus horikoshii in 3 M guanidine hydrochloride.

This study was carried out to investigate the structural perturbation of the protein's local structure by the denaturants under non-denaturing conditions. Crystal structure of CutA from an archaeon Pyrococcus horikosii (PhoCutA), a heavy-metal binding protein, was determined at 1.6-angstroms resolution in the presence of 3 M guanidine HCl (GdnHCl). Native PhoCutA has a large number of short intramolecular hydrogen bonds and salt bridges on the protein surface, of which greater than 90% of hydrogen bonds and all salt bridges were retained in 3 M GdnHCl. Hydrogen bonds that disappeared in the GdnHCl crystal structure were mainly located on the protein surface, especially around the structurally perturbed loop, suggesting interactions between peptide groups and GdnHCl. Only a few GdnH+ ions were observed in the crystal structure, although none at the surface, of the protein. Two GdnH+ ions were observed in the center of the trimeric structure, replacing water molecules, and were hydrogen bonded with Asp84 and Asp86 of each chain. The exterior loop from Tyr39 to Lys44, including Trp40-Trp41, was perturbed structurally. Decreases in temperature factors were observed in beta strand 5 and the N terminus of helix 3. These results suggest the specific bindings of GdnH+ with some acidic residues and the non-specific bindings around Trp residues and peptide groups on the protein surface and that binding of GdnHCl to the native protein is limited, resulting in local structural perturbation.

Archaeal Proteins↗
PubMed2006

Amino acid propensities for secondary structures are influenced by the protein structural class.

Amino acid propensities for secondary structures were used since the 1970s, when Chou and Fasman evaluated them within datasets of few tens of proteins and developed a method to predict secondary structure of proteins, still in use despite prediction methods having evolved to very different approaches and higher reliability. Propensity for secondary structures represents an intrinsic property of amino acid, and it is used for generating new algorithms and prediction methods, therefore our work has been aimed to investigate what is the best protein dataset to evaluate the amino acid propensities, either larger but not homogeneous or smaller but homogeneous sets, i.e., all-alpha, all-beta, alpha-beta proteins. As a first analysis, we evaluated amino acid propensities for helix, beta-strand, and coil in more than 2000 proteins from the PDBselect dataset. With these propensities, secondary structure predictions performed with a method very similar to that of Chou and Fasman gave us results better than the original one, based on propensities derived from the few tens of X-ray protein structures available in the 1970s. In a refined analysis, we subdivided the PDBselect dataset of proteins in three secondary structural classes, i.e., all-alpha, all-beta, and alpha-beta proteins. For each class, the amino acid propensities for helix, beta-strand, and coil have been calculated and used to predict secondary structure elements for proteins belonging to the same class by using resubstitution and jackknife tests. This second round of predictions further improved the results of the first round. Therefore, amino acid propensities for secondary structures became more reliable depending on the degree of homogeneity of the protein dataset used to evaluate them. Indeed, our results indicate also that all algorithms using propensities for secondary structure can be still improved to obtain better predictive results.

Amino Acid Motifs↗
PubMed2003

Structure of Mycobacterium tuberculosis single-stranded DNA-binding protein. Variability in quaternary structure and its implications.

Single-stranded DNA-binding protein (SSB) is an essential protein necessary for the functioning of the DNA replication, repair and recombination machineries. Here we report the structure of the DNA-binding domain of Mycobacterium tuberculosis SSB (MtuSSB) in four different crystals distributed in two forms. The structure of one of the forms was solved by a combination of isomorphous replacement and anomalous scattering. This structure was used to determine the structure of the other form by molecular replacement. The polypeptide chain in the structure exhibits the oligonucleotide binding fold. The globular core of the molecule in different subunits in the two forms and those in Escherichia coli SSB (EcoSSB) and human mitochondrial SSB (HMtSSB) have similar structure, although the three loops exhibit considerable structural variation. However, the tetrameric MtuSSB has an as yet unobserved quaternary association. This quaternary structure with a unique dimeric interface lends the oligomeric protein greater stability, which may be of significance to the functioning of the protein under conditions of stress. Also, as a result of the variation in the quaternary structure the path adopted by the DNA to wrap around MtuSSB is expected to be different from that of EcoSSB.

Amino Acid Sequence↗
PubMed2002

Structural proteomics: developments in structure-to-function predictions.

The major challenge for post-genomic research is to functionally assign and validate a large number of novel target genes and their corresponding proteins. Functional genomics approaches have, therefore, gained considerable attention in the quest to convert this massive data set into useful information. One of the crucial components for the functional understanding of unassigned proteins is the analysis of their experimental or modeled 3D structures. Structural proteomics initiatives are generating protein structures at an unprecedented rate but our current knowledge of 3D-structural space is still limited. Estimates on the completeness of the 3D-structural coverage of proteins vary but it is generally accepted that only a minority of the structural proteome has a template structure from which reliable conclusions can be drawn. Thus, structural proteomics has set out to build a map of protein structures that will represent all protein folds included in the 'global proteome'.

Amino Acid Sequence↗
PubMed1995

Structure and function of cytochromes P450: a comparative analysis of three crystal structures.

BACKGROUND: Cytochromes P450 catalyze the oxidation of a variety of hydrophobic substrates. Sequence identities between P450 families are generally low (10-30%), and consequently, the structure-function correlations among P450s are not clear. The crystal structures of P450terp and the hemoprotein domain of P450BM-3 were recently determined, and are compared here with the previously available structure of P450cam. RESULTS: The topology of all three enzymes is quite similar. The heme-binding core structure is well conserved, except for local differences in the I helices. The greatest variation is observed in the substrate-binding regions. The structural superposition of the proteins permits an improved sequence alignment of other P450s. The charge distribution in the three structures is similarly asymmetric and defines a molecular dipole. CONCLUSIONS: Based on this comparison we believe that all P450s will be found to possess the same tertiary structure. The ability to precisely predict other P450 substrate-contact residues is limited by the extreme structural heterogeneity in the substrate-recognition regions. The central I-helix structures of P450terp and P450BM-3 suggest a role for helix-associated solvent molecules as a source of catalytic protons, distinct from the mechanism for P450cam. We suggest that the P450 molecular dipole might aid in both redox-partner docking and proton recruitment for catalysis.

Amino Acid Sequence↗
PubMed2001

Free-energy calculations highlight differences in accuracy between X-ray and NMR structures and add value to protein structure prediction.

BACKGROUND: While X-ray crystallography structures of proteins are considerably more reliable than those from NMR spectroscopy, it has been difficult to assess the inherent accuracy of NMR structures, particularly the side chains. RESULTS: For 15 small single-domain proteins, we used a molecular mechanics-/dynamics-based free-energy approach to investigate native, decoy, and fully extended alpha conformations. Decoys were all less energetically favorable than native conformations in nine of the ten X-ray structures and in none of the five NMR structures, but short 150 ps molecular dynamics simulations on the experimental structures caused them to have the lowest predicted free energy in all 15 proteins. In addition, a strong correlation exists (r(2) = 0.86) between the predicted free energy of unfolding, from native to fully extended conformations, and the number of residues. CONCLUSIONS: This work suggests that the approximate treatment of solvent used in solving NMR structures can lead NMR model conformations to be less reliable than crystal structures. This conclusion was reached because of the considerably higher calculated free energies and the extent of structural deviation during aqueous dynamics simulations of NMR models compared to those determined by X-ray crystallography. Also, the strong correlation found between protein length and predicted free energy of unfolding in this work suggests, for the first time, that a free-energy function can allow for identification of the native state based on calculations on an extended state and in the absence of an experimental structure.

Crystallography, X-Ray↗
PubMed1992

Recognition of tertiary structure in tRNAs by Rh(phen)2phi3+, a new reagent for RNA structure-function mapping.

With photoactivation Rh(phen)2phi3+ promotes strand cleavage at sites of tertiary interaction in tRNA. The rhodium complex, which binds double-helical DNA by intercalation in the major groove, yields no cleavage in double-helical regions of the RNA or in unstructured single-stranded regions. Instead, Rh(phen)2phi3+ appears to target regions which are structured so that the major groove is open and accessible for stacking with the complex, as occurs where bases are triply bonded. So as to examine the specificity of this novel reagent and to evaluate its use in probing structural changes in RNAs, cleavage studies have been conducted on two structurally characterized tRNAs, tRNA(Phe) and tRNA(Asp) from yeast, the unmodified yeast tRNA(Phe) transcript, and a chemically modified tRNA(Phe), as well as on a series of tRNA(Phe) mutants. On tRNA(Phe) strong cleavage is observed at residues G22, G45, U47, psi 55, and U59; weaker cleavage is observed at A44, m7G46, and C48. On tRNA(Asp) cleavage is found at residues A21 through G26, psi 32, and U48, with minor cleavage apparent at A44, G45, A46, psi 55, U59, and U60. There is a striking similarity in cleavage observed on these tRNAs, and the sites of cleavage mark regions of tertiary folding. Cleavage on the unmodified tRNA(Phe) transcript resembles closely that found on native yeast tRNA(Phe), but additional sites, primarily in the anticodon loop and stem, are evident. The results indicate that globally the structures containing or lacking the modified bases appear to be the same; the differences in cleavage observed may reflect a loosening or alteration in the structure due to the absence of the modified bases. Cleavage results on mutants of tRNA(Phe) illustrate Rh(phen)2phi3+ as a sensitive probe in characterizing tRNA tertiary structure. Results are consistent with other assays for structural or functional changes. Uniquely, Rh(phen)2phi3+ appears to target directly sites of tertiary interaction. Cleavage results on mutants which involve base changes within the triply bounded region of the molecule indicate that it is the structure of the triply bonded array rather than the individual nucleotides which are being targeted. Chemical modification to promote selective depurination of the third base (m7G46) involved in the triple in the folded, native tRNA leads to the reduction of cleavage by the metal complex; this result shows directly the importance of the stacked triple base structure for recognition by the metal complex.(ABSTRACT TRUNCATED AT 400 WORDS)

2,2'-Dipyridyl↗
PubMed1992

1.67-A X-ray structure of the B2 immunoglobulin-binding domain of streptococcal protein G and comparison to the NMR structure of the B1 domain.

The structure of the B2 immunoglobulin-binding domain of streptococcal protein G has been determined at 1.67-A resolution using a combination of single isomorphous replacement (SIR) phasing and manual fitting of the coordinates of the NMR structure of B1 domain of streptococcal protein G [Gronenborn, A. M., et al. (1991) Science 253, 657-661]. The final R value was 0.191 for data between 8.0 and 1.67 A. The structure described here has 13 residues preceding the 57-residue Ig-binding domain and 13 additional residues following it, for a total of 83 residues. The 57-residue binding domain is well-determined in the structure, having an average B factor of 18.0. Only residues 8-77 could be located in the electron density maps, with the ends of the structure fading into disorder. Like the B1 domain, the B2 domain consists of four beta-strands and a single helix lying diagonally across the beta-sheet, with a -1, +3 chi, -1 topology. This small structure is extensively hydrogen-bonded and has a relatively large hydrophobic core. These structural observations may account for the exceptional stability of protein G. A comparison of the B2 domain X-ray structure and the B1 domain NMR structure showed minor differences in the turn between strands and two and a slight displacement of the helix relative to the sheet. Hydrogen bonds between crystallographically related molecules account for most of these differences.

Amino Acid Sequence↗
PubMed1993

Structure and thermodynamics of nonalternating C.G base pairs in Z-DNA: the 1.3-A crystal structure of the asymmetric hexanucleotide d(m5CGGGm5CG).d(m5CGCCm5CG).

We have solved the single-crystal X-ray structure of the complementary hexanucleotides d(m5-CGGGm5CG) and d(m5CGCCm5CG). The hexamer duplex was crystallized as Z-DNA, but contains a single C.G base pair that does not follow the alternating pyrimidine/purine rule for Z-DNA formation. This is the first crystal structure which serves to illustrate the structural consequences of placing a cytosine in the sterically disfavored syn conformation. In addition, since these sequences are not self-complementary, the individual strands of this asymmetric hexamer are unique in sequence and therefore distinguishable in the crystal lattice. Nevertheless, the crystal of this duplex is isomorphous with other Z-DNA hexamer structures. The asymmetry of this hexamer sequence required that the structure be solved using two unique models, which are distinguished by the orientation of hexanucleotides in the crystal lattice. In one model (the GG model) the cytosine in the syn conformation is packed against the terminal guanine base of a symmetry-related hexamer, while in the alternative model (the CC model) this cytosine sits exposed in a solvent channel of the lattice. We find that neither model alone can completely account for the observed electron densities. The two models ultimately were refined together. A composite structure consisting of 65% GG model and 35% CC model refined to an R-factor of 19.3%, which was significantly lower than refinements using either model alone. A detailed analysis of these two structures shows that, in spite of the out-of-alternation C.G base pair, the features characteristic of Z-DNA have been maintained. Both models, however, show significant local structural adjustments to accommodate the single cytosine base which is forced to adopt the syn conformation in each hexamer. In general, it appears that in order to relieve the energetically unfavorable steric contacts between the cytosine base in the syn conformation and the deoxyribose sugar, the base is forced into a highly buckled conformation, and that this large buckle in turn alters the conformation of neighboring residues. This unusual conformation also significantly weakens base-stacking interactions between the cytosine in syn and the adjacent residues in the helix and affects the exposure of the bases to solvent. We conclude that this crystal structure provides a molecular rationale for why nonalternating bases are energetically disfavored in Z-DNA.

Base Composition↗
PubMed1989

Solution structure of the EcoRI DNA sequence: refinement of NMR-derived distance geometry structures by NOESY spectrum back-calculations.

The solution structure of the self-complementary DNA duplex [d(CGCGAATTCGCG)]2, which contains the EcoRI restriction site sequence GAATTC at the center, has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Time-dependent nuclear Overhauser effect spectra were used to obtain the initial cross-relaxation rates between 155 pairs of protons. These initial cross-relaxation rates were converted into interproton distances and entered into a distance (bounds) matrix. A distance geometry algorithm (DSPACE) was used to create embedded starting structures and to refine these structures until they showed good agreement with the distance matrix; symmetry constraints were included in the refinement procedure, making the two strands in the refined distance geometry structures virtually identical and significantly improving the agreement with the distance matrix. The NOESY spectrum for one of these distance geometry structures was then calculated from the explicit coordinates by numerically integrating all the z-magnetization transfer pathways among neighboring protons within a specified radius. Distances in this distance geometry structure that did not agree with the experimental NOESY time course were then adjusted accordingly. This process was iterated until a good agreement between calculated and experimental NOESY spectra was reached. The final structure, which generates good agreement with the experimental NOESY spectrum, displays kinks at the C3-G4 base step and at the A6-T7 base step that appear to be similar to those reported for the EcoRI restriction site DNA bound to its endonuclease. The solution structure is not the same as the crystal structure of this DNA duplex.

Base Sequence↗
PubMed2004

1H NMR characterization of the solution active site structure of substrate-bound, cyanide-inhibited heme oxygenase from Neisseria meningitidis: comparison to crystal structures.

Heme oxygenase, HO, from the pathogenic bacterium Neisseria meningitidis catabolizes heme for the iron necessary for infection. The enzyme, labeled HemO, exhibits less sequence homology to mammalian HO than another studied HO from Corynebacterium diphtheriae. Solution 1H NMR has been utilized to define the active site molecular and electronic structure of the cyanide-inhibited, substrate-bound complex for comparison with those provided by several crystal structures. Extensive assignments by solely 1H NMR 2D methods reveal a structure that is very strongly conserved with respect to the crystal structure, although 1H/2H exchange indicates dynamically much more stable distal and proximal helices than those for other HOs. Several residues found with alternate orientations in crystal structures of water- and NO-ligated complexes were shown to occupy positions found solely in the NO complex, confirming that there are structural accommodations in response to ligating the substrate complex with a diatomic, H-bond acceptor ligand. The observed dipolar shifts allow the determination of the magnetic axes that show that the Fe-CN unit is tilted approximately 10 degrees toward the alpha-meso position, thereby facilitating the alpha-stereoselectivity of the enzyme. Numerous labile protons with larger than usual low-field bias are identified and, in common with the other HO complexes, shown to participate in an extended, distal side H-bond network. This H-bond network orders several water molecules, most, but not all, of which have been detected crystallographically. A series of three C-terminal residues, His207-Arg208-His209, are not detected in crystal structures. However, 1H NMR finds two residues, His207 and likely Arg208 in contact with pyrrole D, which in crystal structures is exposed to solvent. The nature of the NOEs leads us to propose a H-bond between the proximally oriented His207 ring and the carboxylate of Asp27 and a salt-bridge between the terminus of Arg208 and the reoriented 7-propionyl carboxylate. While numerous ordered water molecules are found near both propionates in the crystal structure, we find much larger water NOEs to the 6- than 7-propionate, suggesting that water molecules near the 7-propionate have been expelled from the cavity by the insertion of Arg208 into the distal pocket. The conversion of the 7-propionate link from the N-terminal region (Lys16) to the C-terminal region (Arg208) in the ligated substrate complex both closes the heme cavity more tightly and may facilitate product exit, the rate-limiting step in the enzyme activity.

Amino Acid Sequence↗
PubMed1996

Chloroplastic aspartate aminotransferase from Arabidopsis thaliana: an examination of the relationship between the structure of the gene and the spatial structure of the protein.

A clone encoding a plastid isoenzyme of aspartate amino-transferase (AAT5) was isolated from an Arabidopsis genomic library and its complete sequence determined. The gene for AAT5 (asp5) contains an open reading frame of 2447 bp comprising 11 exons separated by introns ranging in length from 74 to 207 bp. The upstream regulatory region contains a putative TATA box and multiple copies of two sequence motifs, CTCTT and AAAGAT, previously associated with nodule-specific gene activity in legumes. The deduced primary amino acid sequence of the protein product of asp5 was used to generate a three-dimensional structure of the AAT5 protein by using the computer program Sybyl: Biopolymer Composer and known AAT structures on the protein databases. Both the mature protein and its precursor protein containing a putative N-terminal transit peptide were modelled. The resulting structure of the precursor protein indicated that the transit peptide might also inhibit dimerization of the protein until after its translocation across the chloroplast membrane. The derived structure of the mature protein was then analysed in terms of its component elements of secondary structure, and the positions on the polypeptide back-bone corresponding to intron insertion sites were determined. It is observed that the introns tend to map to regions between structural subdomains of the protein and also map to sites on the surface of the molecule. The asp5 gene in Arabidopsis is thus consistent with Gilbert's exon-shuffling theory of gene evolution [Gilbert (1985) Science 228, 823-824]. A high degree of conservation of intron insertion sites between AAT genes from different plants and animals is observed, particularly within the part of the gene encoding a large beta-sheet structure that forms the structural and functional core of the protein. This beta-sheet structure is thus believed to compromise an ancient and very highly conserved moiety of the molecule.

Amino Acid Sequence↗