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At least 397 records · Page 22Linked to original sources
PubMed1993

Representing an ensemble of NMR-derived protein structures by a single structure.

The usefulness of representing an ensemble of NMR-derived protein structures by a single structure has been investigated. Two stereochemical properties have been used to assess how a single structure relates to the ensemble from which it was derived, namely the distribution of phi psi torsion angles and the distribution of chi 1 torsion angles. The results show that the minimized average structure derived from the ensemble (a total of 11 ensembles from the Brookhaven Protein Data Bank were analyzed) does not always correspond well with this ensemble, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue. An alternative method that selects the member of the ensemble which is closest to the "average" of the ensemble has been investigated (a total of 23 ensembles from the Brookhaven Protein Data Bank were analyzed). Although this method selected a structure that on the whole corresponded more closely to the ensemble than did the minimized average structure, this is still not a totally reliable means of selecting a single structure to represent the ensemble. This suggests that it is advisable to study the ensemble as a whole. A study has also been made of the practice of selecting the "best" rather than the most representative member of the ensemble. This too suggests that the ensemble should be studied as a whole. A study of the conformational space occupied by the ensemble also suggests the need to consider the ensemble as a whole, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue.

Animals↗
PubMed1996

Improving the quality of NMR and crystallographic protein structures by means of a conformational database potential derived from structure databases.

A new conformational database potential involving dihedral angle relationships in databases of high-resolution highly refined protein crystal structures is presented as a method for improving the quality of structures generated from NMR data. The rationale for this procedure is based on the observation that uncertainties in the description of the nonbonded contacts present a key limiting factor in the attainable accuracy of protein NMR structures and that the nonbonded interaction terms presently used have poor discriminatory power between high- and low-probability local conformations. The idea behind the conformational database potential is to restrict sampling during simulated annealing refinement to conformations that are likely to be energetically possible by effectively limiting the choices of dihedral angles to those that are known to be physically realizable. In this manner, the variability in the structures produced by this method is primarily a function of the experimental restraints, rather than an artifact of a poor nonbonded interaction model. We tested this approach with the experimental NMR data (comprising an average of about 30 restraints per residue and consisting of interproton distances, torsion angles, 3JHN alpha coupling constants, and 13C chemical shifts) used previously to calculate the solution structure of reduced human thioredoxin (Qin J, Clore GM, Gronenborn AM, 1994, Structure 2:503-522). Incorporation of the conformational database potential into the target function used for refinement (which also includes terms for the experimental restraints, covalent geometry, and nonbonded interactions in the form of either a repulsive, repulsive-attractive, or 6-12 Lennard-Jones potential) results in a significant improvement in various quantitative measures of quality (Ramachandran plot, side-chain torsion angles, overall packing). This is achieved without compromising the agreement with the experimental restraints and the deviations from idealized covalent geometry that remain within experimental error, and the agreement between calculated and observed 1H chemical shifts that provides an independent NMR parameter of accuracy. The method is equally applicable to crystallographic refinement, and should be particular useful during the early stages of either an NMR or crystallographic structure determination and in cases where relatively few experimental restraints can be derived from the measured data (due, for example, to broad lines in the NMR spectra or to poorly diffracting crystals).

Computer Simulation↗
PubMed1990

Crystal structures of subtilisin BPN' variants containing disulfide bonds and cavities: concerted structural rearrangements induced by mutagenesis.

The X-ray structures of four genetically engineered disulfide variants of subtilisin have been analyzed to determine the energetic and structural constraints involved in inserting disulfide bonds into proteins. Each of the engineered disulfides exhibited atypical sets of dihedral angles compared with known structures of natural disulfide bridges in proteins and affected its local structural environment to a different extent. The disulfides located in buried regions, Cys26-Cys232 and Cys29-Cys119, induced larger changes than did Cys24-Cys87 and Cys22-Cys87, which are located on the surface of the molecule. An analysis of the concerted changes in secondary structure units such as alpha-helices and beta-sheets indicated systematic long-range effects. The observed changes in the mutants were largely distributed asymmetrically around the inserted disulfides, reflecting different degrees of inherent flexibility of neighboring secondary structure types. The disulfide substitution in each variant molecule created some invaginations or cavities, causing a reorganization of the surrounding water structure. These changes are described, as well as the changes in side chain positions of groups that border the cavities.

Base Sequence↗
PubMed1996

Refined structure of lac repressor headpiece (1-56) determined by relaxation matrix calculations from 2D and 3D NOE data: change of tertiary structure upon binding to the lac operator.

The solution structure of the DNA binding domain of lac repressor (headpiece 1-56; HP56) has been refined using data from 2D and 3D NMR spectroscopy. The structure was derived from 1546 restraints (giving an average of 27.6 per residue), comprising 389 intraresidual, 402 sequential, 385 medium range and 325 long range distance restraints and also 30 phi and 15 chi 1 dihedral angle restraints. The structures were determined by the method of direct refinement against nuclear Overhauser enhancement peak volumes with the program DINOSAUR. The final set of 32 selected structures displayed an r.m.s. deviation from the average of 0.43(+/-0.08) A angstroms (backbone) and 0.95(+/-0.08) angstroms (all heavy atoms) for the best defined region of the protein (residues 3 to 49). The ensemble R-factor was 0.35, which indicates close correspondence with the experimental data. The structures revealed good stereochemical qualities. The conformations of the NMR structures of free and DNA complexed lac repressor headpiece were compared. The regions comprising the secondary structure elements show close correspondence for both conformations. However, the conformation of the loop between helix II and III changes considerably upon complexation of the headpiece. This change in the conformation of the loop in lac HP56 is essential for binding of the side-chains of residues Asn25 and His29 to the lac operator DNA. Finally, the lac headpiece residues that are intolerant to mutations were analysed. Most of these mutation-sensitive residues are important for a correct folding of the headpiece region, and a number of these residues are also involved in contacting the operator DNA.

Crystallography, X-Ray↗
PubMed1998

Structural and functional roles of heme binding module in globin proteins: identification of the segment regulating the heme binding structure.

To investigate structural and functional significance of a newly proposed structural unit in globins, the "heme binding module", we synthesized a "heme binding module"-substituted chimeric globin and characterized its function and structure. In our previous study we proposed that the heme binding module, corresponding to the segment from Leu(F1) to Phe(G5) in hemoglobin alpha-subunit, plays a key role in constructing the heme proximal structure in globins. The replacement of the heme binding module in myoglobin with that of hemoglobin alpha-subunit converted the absorption spectra into that of the alpha-subunit, and, in the resonance Raman spectra, the vibration mode characteristic of myoglobin completely disappeared after the module replacement. The hyperfine-shifted NMR resonances for the cyanide-bound form of the module-substituted myoglobin also revealed that the orientation of the axial histidine is close to that of the alpha-subunit rather than that of myoglobin, while the deviations of the resonance positions of the NMR signals from the amino acid residues located in the distal site were subtle, supporting the preferential structural alterations in the heme proximal site. The present finding for the structural alterations in the module-substituted myoglobin confirms that the heme binding module can be a segment regulating the heme proximal structure in globin proteins.

Amino Acid Sequence↗
PubMed2000

An integrated approach to the analysis and modeling of protein sequences and structures. II. On the relationship between sequence and structural similarity for proteins that are not obviously related in sequence.

Here, we discuss the relationship between protein sequence and protein structural similarity. It is established that a protein structural distance (PSD) of 2.0 is a threshold above which two proteins are unlikely to have a detectable pairwise sequence relationship. A precise correlation is established between the level of sequence similarity, defined by a normalized Smith-Waterman score, and the probability that two proteins will have a similar structure (defined by pairwise PSD<2). This correlation can be used in evaluating the likelihood for success in a comparative modeling procedure. We establish the existence of a correlation between sequence and structural similarity for pairs of proteins that are related in structure but whose sequence relationship is not detectable using standard pairwise sequence alignments. Although it is well known that there is a close relationship between sequence and structural similarity for pairwise sequence identities greater than about 30 %, there has been little discussion as to the possible existence of such a relationship for pairs of proteins in or below the twilight zone of sequence similarity (<25 % pairwise sequence identity). Possible implications of our results for the evolution of protein structure are discussed.

Algorithms↗
PubMed2001

Contribution of peptide bonds to inhibitor-protease binding: crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3-Pro18I and OMTKY3-psi[COO]-Leu18I in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY-3-psi[CH2NH2+]-Asp19I.

X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3). The natural P1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-psi[COO]-Leu18I). Both variants lack the P1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O(gamma) of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors. The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the DeltaG degrees of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-psi[COO]-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P1 NH group. Therefore, the difference in DeltaG degrees, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P1 NH hydrogen bond toward binding. A crystal structure of OMTKY3 having a reduced peptide bond between P1 Leu18I and P'1 Asp19I, (OMTKY3-psi[CH2NH2+]-Asp19I) has also been determined by X-ray crystallography. This variant has very weak association equilibrium constants with SGPB and with chymotrypsin. The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole.

Animals↗
PubMed1991

Structure determination of the Antp (C39----S) homeodomain from nuclear magnetic resonance data in solution using a novel strategy for the structure calculation with the programs DIANA, CALIBA, HABAS and GLOMSA.

The structure of a mutant Antennapedia homeodomain, Antp(C39----S), from Drosophila melanogaster was determined using a set of new programs introduced in the accompanying paper. An input dataset of 957 distance constraints and 171 dihedral angle constraints was collected using two-dimensional n.m.r. experiments with the 15N-labeled protein. The resulting high quality structure for Antp(C39----S), with an average root-mean-square deviation of 0.53 A between the backbone atoms of residues 7 to 59 in 20 energy-refined distance geometry structures and the mean structure, is nearly identical to the previously reported structure of the wild-type Antp homeodomain. The only significant difference is in the connection between helices III and IV, which was found to be less kinked than was indicated by the structure determination for Antp. The main emphasis of the presentation in this paper is on a detailed account of the practical use of a novel strategy for the computation of nuclear magnetic resonance structures of proteins with the combined use of the programs DIANA, CALIBA, HABAS and GLOMSA.

Algorithms↗
PubMed1994

Principles determining the structure of beta-sheet barrels in proteins. II. The observed structures.

In the accompanying paper we derived a set of principles that, we argue, govern the structure of beta-sheet barrels. Barrel structures are classified in terms of two integral parameters: the number of strands in the beta-sheet, n, and a measure of the stagger in the beta-sheet, S. We derived a set of equations that show how the (n, S) values of a barrel structure determine the arrangement of its strands; its general shape; the twist and coiling of the beta-sheet, and the arrangement of residues in the barrel interior. This work suggested that there are ten different combinations of n and S that form barrels with good beta-sheet geometries and interiors close packed by beta-sheet residues. In this paper we demonstrate the validity of these principles. We analyse in detail the observed structures of 39 different beta-sheet barrels. These structures include representatives of all the different barrel structures currently known and for which atomic co-ordinates are available. We show that the observed arrangement of the strands, and the extent of the twist and coiling of the beta-sheets, are very close to those calculated from the (n, S) values for the barrel. Of the 39 structures, 34 have one of the ten (n, S) values that we expect to form barrels with good beta-sheet geometries and interiors close packed by beta-sheet residues. The other five have one of two (n, S) values that give good beta-sheet geometries but radii so large the beta-sheet residues leave cavities at the centre of the barrels. In at least four of these cavities have a functional role.

Amino Acid Sequence↗
PubMed2004

Structural roles of the active site iron(III) ions in catechol 1,2-dioxygenases and differential secondary structure changes in isoenzymes A and B from Acinetobacter radioresistens S13.

The reversible active site metal ion removal process for two catechol 1,2-dioxygenase isoenzymes (IsoA and IsoB) isolated from Acinetobacter radioresistens S13 has been monitored using circular dichroism and fluorescence spectroscopic techniques. IsoA and IsoB are homodimers, containing one iron(III) ion per subunit. Their amino acid sequence identity is 48.4%. Previous experiments suggested that structural diversities could be responsible for the differential thermal and pH stabilities of the two isoenzymes and of their distinct demetallation kinetics. The far-UV CD spectra of IsoA and IsoB catechol 1,2-dioxygenases from A. radioresistens S13 provide information on their secondary structures. IsoB appears to have a content of alpha-helices higher than IsoA. Upon metal ion removal, both proteins reversibly lose part of their secondary structure following distinct pathways. CD spectra simulations allowed us to estimate the content of alpha-helices, beta-sheets, and turns for each isoenzyme and to monitor the secondary structure rearrangements. The metal ion withdrawal has large influence on the secondary structure: in particular a significant reduction of alpha-helices content is observed for both isoenzymes. Intrinsic fluorescence emission spectra clearly support such results, adding information on the local environment changes of the tryptophan residues. The positioning of Trp250 in IsoB has been shown to be of particular interest for monitoring the local structure changes occurring upon metal ion removal. For the first time these studies allow to underline the role of active site iron ions on dioxygenases folding and stability, further evidencing the differences in structural assembling between the two isoenzymes from A. radioresistens S13.

Acinetobacter↗
PubMed2005

1.42A crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure.

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1.

Binding Sites↗
PubMed2000

Solution structure of the loops of bacteriorhodopsin closely resembles the crystal structure.

Bacteriorhodopsin is one of very few transmembrane proteins for which high resolution structures have been solved. The structure shows a bundle of seven helices connected by six turns. Some turns in proteins are stabilized by short range interactions and can behave as small domains. These observations suggest that peptides containing the sequence of the turns in a membrane protein such as bacteriorhodopsin may form stable turn structures in solution. To test this hypothesis, we determined the solution structure of three peptides each containing the sequence of one of the turns in bacteriorhodopsin. The solution structures of the peptides closely resemble the structures of the corresponding turns in the high resolution structures of the intact protein.

Amino Acid Sequence↗
PubMed1997

Crystal structure of the chemotaxis receptor methyltransferase CheR suggests a conserved structural motif for binding S-adenosylmethionine.

BACKGROUND: Flagellated bacteria swim towards favorable chemicals and away from deleterious ones. The sensing of chemoeffector gradients involves chemotaxis receptors, transmembrane proteins that detect stimuli through their periplasmic domains and transduce signals via their cytoplasmic domains to the downstream signaling components. Signaling outputs from chemotaxis receptors are influenced both by the binding of the chemoeffector ligand to the periplasmic domain and by methylation of specific glutamate residues on the cytoplasmic domain of the receptor. Methylation is catalyzed by CheR, an S-adenosylmethionine-dependent methyltransferase. CheR forms a tight complex with the receptor by binding a region of the receptors that is distinct from the methylation site. CheR belongs to a broad class of enzymes involved in the methylation of a variety of substrates. Until now, no structure from the class of protein methyltransferases has been characterized. RESULTS: The structure of the Salmonella typhimurium chemotaxis receptor methyltransferase CheR bound to S-adenosylhomocysteine, a product and inhibitor of the methylation reaction, has been determined at 2.0 A resolution. The structure reveals CheR to be a two-domain protein, with a smaller N-terminal helical domain linked through a single polypeptide connection to a larger C-terminal alpha/beta domain. The C-terminal domain has the characteristics of a nucleotide-binding fold, with an insertion of a small antiparallel beta sheet subdomain. The S-adenosylhomocysteine-binding site is formed mainly by the large domain, with contributions from residues within the N-terminal domain and the linker region. CONCLUSIONS: The CheR structure shares some structural similarities with small molecule DNA and RNA methyltransferases, despite a lack of sequence similarity among them. In particular, there is significant structural preservation of the S-adenosylmethionine-binding clefts; the specific length and conformation of a loop in the alpha/beta domain seems to be required for S-adenosylmethionine binding within these enzymes. Unique structural features of CheR, such as the beta subdomain, are probably necessary for CheR's specific interaction with its substrates, the bacterial chemotaxis receptors.

Amino Acid Sequence↗
PubMed1995

Solution structure of the cytoplasmic domain of phopholamban: phosphorylation leads to a local perturbation in secondary structure.

Peptides representing the N-terminal domain (Ia) of the cardiac sarcoplasmic reticulum protein phospholamban (residues 1-25 [PLB(1-25)] and a phosphorylated form [pPLB(1-25)]) were synthesized and their conformations examined using circular dichroism and nuclear magnetic resonance spectroscopy. In aqueous solution, both PLB(1-25) and pPLB(1-25) adopt a primarily disordered conformation. In 30% trifluoroethanol/10 mM phosphate, PLB(1-25) exhibits a CD spectrum consistent with 60% helical structure. This value decreases to 27% for the phosphorylated peptide. CD spectra in 2% SDS indicate 40% alpha-helix for PLB(1-25) and 20% for pPLB(1-25). Full chemical shift assignments were obtained by conventional homonuclear NMR methodologies for both PLB(1-25) and pPLB(1-25) in 30% trifluoroethanol/water and 300 mM SDS. The solution structure of PLB(1-25) in 30% TFE/water was determined from distance geometry calculations using 54 NOE distance constraints and 17 torsion angle constraints. In the family of 20 calculated conformers, the root mean square deviation from the mean structure is 0.79 A for backbone heavy atoms of residues 1-17. The structure comprises a regular alpha-helix extending from M1 to S16 with the remaining C-terminal residues disordered. The calculated structure is supported by analysis of C alpha H secondary shifts which are significantly negative for residues 1-16. Chemical shift degeneracy is substantially more extensive in the phospho form and precludes a direct comparison of calculated structures. However, the magnitudes of upfield secondary shifts are decreased by 20% in residues 1-11 and are not significantly helical for residues 12-16 according to the criteria of Wishart et al. [(1992) Biochemistry 31, 1647-1651]. 3JHN alpha coupling constants measured for I12, R13, A15, and S16 also suggest that residues 12-16 undergo a local unwinding of the helix upon phosphorylation. Similar results are obtained for PLB(1-25) and pPLB(1-25) in 300 mM perdeuterated sodium dodecyl sulfate except that differences in backbone dynamics for the helical and nonhelical regions of the peptide are evident in the DQF-COSY line shapes for fingerprint cross-peaks. This disruption of structure at the C-terminus of the helix suggests a model for phosphorylation-induced dissociation of the PLB/Ca(2+)-ATPase complex.

Amino Acid Sequence↗
PubMed1992

Refinement of the crystal structure of ribonuclease S. Comparison with and between the various ribonuclease A structures.

Ribonuclease S (RNase-S) is a complex that consists of two proteolytic fragments of bovine pancreatic ribonuclease A (RNase-A): the S-peptide (residues 1-20) and S-protein (residues 21-124). We have refined the crystal structures of three RNase-S complexes. The first two contain the full-length 20-residue S-peptide and were studied at pHs of 4.75 and 5.5. The third one consists of a truncated form of S-peptide (residues 1-15) and was studied at pH 4.75 as the reference structure for a series of mutant peptide complexes to be reported separately. Excluding residues 16-23 which are either missing (in the S15 complex) or disordered (in both S20 complexes), all three structures refined at 1.6-A resolution are identical within the estimated errors in the coordinates (0.048 A for the backbone atoms). The R-values, residual error, range from 17.4% to 18.6%. The final model of S20, pH 4.75, includes 1 sulfate and 84 water molecules. The side chains of 11 residues were modeled in two discrete conformations. The final structures were independent of the particular RNase-A or RNase-S used as a starting model. An extensive comparison with refined crystal structures of RNase-A reveals that the core of the molecule which is held together with extensive hydrogen bonds is in identical pattern in all cases. However, the loop regions vary from one structure to another and are often characterized by high B-factors. The pattern of thermal parameters appears to be dependent on crystal packing and correlates well with the accessibility calculated in the crystal. Gln60 is a conserved residue in all sequences known to date for this class of ribonucleases. However, it is the only residue that is clearly defined in an unfavorable position (phi = -100 degrees, psi = -130 degrees) on the Ramachandran plot. The origin of the substantial differences between RNase-A and RNase-S in stability to both acid and temperature denaturation and in susceptibility to proteolysis at neutral pH is not obvious in our visual comparison of these two structures.

Animals↗
PubMed1994

Secondary structure of the nicotinic acetylcholine receptor: implications for structural models of a ligand-gated ion channel.

The secondary structure and effects of two ligands, carbamylcholine and tetracaine, on the secondary structure of affinity-purified nicotinic acetylcholine receptor (nAChR) from Torpedo has been studied using Fourier transform infrared spectroscopy (FTIR). FTIR spectra of the nAChR were acquired in both 1H2O and 2H2O buffer and exhibit spectral features indicative of a substantial alpha-helical content with lesser amounts of beta-sheet and random coil structures. The resolution enhancement techniques of Fourier self-deconvolution and Fourier derivation reveal seven component bands contributing to both the amide I band and amide I' band contours in 1H2O and 2H2O, respectively. Curve-fitting estimates of the nAChR secondary structure are consistent with the qualitative analysis of the FTIR spectra as follows: 39% alpha-helix, 35% beta-sheet, 6% turn, and 20% random coil. Of particular interest is the estimated alpha-helical content as this value places restrictions on models of the nAChR transmembrane topology and on the types of secondary structures that may contribute to functional domains, such as the ligand-binding site. The estimated alpha-helical content is sufficient to account for four transmembrane alpha-helices in each nAChR subunit as well as a substantial portion of the extracellular and/or the cytoplasmic domains. FTIR spectra were also acquired in the presence and absence of 1 mM carbamylcholine and 5 mM tetracaine to examine the effects of ligand binding on the secondary structure of the nAChR. The similarity of the spectra, even after spectral deconvolution, indicates that the secondary structure of the nAChR is essentially unaffected by desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Solution structure of a monoheme ferrocytochrome c from Shewanella putrefaciens and structural analysis of sequence-similar proteins: functional implications.

Within the frame of the characterization of the structure and function of cytochromes c, an 81-amino acid cytochrome c was identified in the genome of Shewanella putrefaciens. Because of the scarce information about bacterial cytochromes of this type and the large variability in sequences and possibly function, we decided to proceed to its structural characterization. This protein was expressed in Escherichia coli and purified. The oxidized species is largely high spin, with a detached methionine, whereas the reduced species has the classical His/Met axial ligation to iron. The NMR solution structure of the reduced form was determined on a (15)N-labeled sample, for which 99% of all non-proline backbone (1)H and (15)N resonances have been assigned. One thousand three hundred two meaningful NOEs, out of 1775 NOEs, together with 66 dihedral angles provide a structure with rmsd values from the mean of 0.50 and 0.96 A for backbone and all heavy atoms, respectively. A search of gene banks allowed us to locate 10 different cytochromes c, the sequences of which are more than 30% identical to that of the S. putrefacienscytochrome. For two of them, the structures are known. The structures of the others have been modeled by using the available templates and internally validated. Structural similarities in terms of surface properties account for their biophysical features and provide hints about the function.

Amino Acid Sequence↗
PubMed1998

Tertiary structure of the major house dust mite allergen Der p 2: sequential and structural homologies.

Sensitization to indoor allergens, especially those of the house dust mite, is strongly correlated with the development of asthma. We report the tertiary structure of the major house dust mite allergen, Der p 2, determined by NMR methods. The structure of Der p 2 is a beta-barrel and is composed of two three-stranded antiparallel beta-pleated sheets. This arrangement of beta-strands is similar to the immunoglobulin fold with respect to the orientation of the two sheets and the interactions of the strands. However, the three-dimensional structure of Der p 2 aligns equivalently with a number of proteins from different families within the immunoglobulin superfamily. The structural homology with the highest significance score from analysis by DALI is to Der f 2. Although Der p 2 and Der f 2 are 87% identical in amino acid sequence, they align in three dimensions rather poorly (4.85 A RMSD; Z-score, 8.58). This unexpected finding is likely due to the different solution conditions used during structure determination by NMR for both proteins. While the structural comparisons did not elucidate a clear homologue for the function of Der p 2 in mites, we report that Der p 2 is sequentially homologous to esr16. This is a protein from moths that is expressed coincident with molting. Thus, this homology has important ramifications for the study of mite allergy. The structure of Der p 2 provides a useful tool in the design of recombinant immunotherapeutics for the group 2 allergens.

Allergens↗