bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering the substrate specificity of rhizopuspepsin: The role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1
Authors: Todd Lowther W. , Dunn Ben M. , Majer Pavel 
Published: June 2010

AbstractRhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild‐type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild‐type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol−1 of transition‐state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1‐lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients.
Secondary and tertiary structure of the A‐state of cytochromecfrom resonance Raman spectroscopy
Authors: Jordan Trace , Eads Janina C. , Spiro Thomas G. 
Published: June 2010

AbstractFerricytochromeccan be converted to the partially folded A‐state at pH 2.2 in the presence of 1.5 M NaCl. The structure of the A‐state has been studied in comparison with the native and unfolded states, using resonance Raman spectroscopy with visible and ultraviolet excitation wavelengths. Spectra obtained with 200 nm excitation show a decrease in amide II intensity consistent with loss of structure for the 50s and 70s helices. The 230‐nm spectra contain information on vibrational modes of the single Trp 59 side chain and the four tyrosine side chains (Tyr 48, 67, 74, and 97). The Trp 59 modes indicate that the side chain remains in a hydrophobic environment but loses its tertiary hydrogen bond and is rotationally disordered. The tyrosine modes Y8b and Y9a show disruption of tertiary hydrogen bonding for the Tyr 48, 67, and 74 side chains. The high‐wavenumber region of the 406.7‐nm resonance Raman spectrum reveals a mixed spin heme iron atom, which arises from axial coordination to His 18 and a water molecule. The low‐frequency spectral region reports on heme distortions and indicates a reduced degree of interaction between the heme and the polypeptide chain. A structural model for the A‐state is proposed in which a folded protein subdomain, consisting of the heme and the N‐terminal, C‐terminal, and 60s helices, is stabilized through nonbonding interactions between helices and with the heme.
Identification of the binding and activating sites of the sphingolipid activator protein, saposin C, with glucocerebrosidase
Authors: Weiler Solly , Tomich John M. , Kishimoto Yasuo , O'Brien John S. , Barranger John A. 
Published: June 2010

AbstractSaposin C is a sphingolipid activator protein of 8.5 kDa that activates lysosomal glucocerebrosidase. Previously, we synthesized and characterized a synthetic full‐length human saposin C protein that displays 85% of the activity of the native saposin C. In this study we use shorter synthetic peptides derived from the saposin C sequence to map binding and activation sites. By determining the activity and kinetic constant (Kact) values of these peptides, we have identified two functional domains, each comprising a binding site adjacent to or partially overlapping with an activation site. Domains 1 and 2 are located within amino acid positions 6–34 and 41–60, respectively. The activation sites span residues 27–34 and 41–49, whereas binding sites encompass residues 6–27 and 45–60. Peptides containing the sequences of either domain displayed 90% of the activity of the full‐length synthetic saposin C. Domain 2, however, bound to glucocerebrosidase by at least an order of magnitude more strongly than domain 1. Binding sites within these domains contain sequences that are excellent candidates for forming amphipathic helical structures. Competition assays demonstrated that the binding of one domain to glucocerebrosidase prevents binding of the other domain, and that saposin A and saposin C bind to the same sites on glucocerebrosidase. A model predicting a saposin C:glucocerebrosidase complex with a stoichiometry of 4:2, respectively, is presented.
Human S100b protein: Formation of a tetramer from synthetic calcium‐binding site peptides
Authors: Donaldson Craig , Barber Kathryn R. , Shaw Gary S. , Kay Cyril M. 
Published: June 2010

AbstractHuman brain S100b protein is a unique calcium‐binding protein comprised of two identical 91‐amino acid polypeptide chains that each contain two proposed helix‐loop‐helix (EF‐hand) calcium‐binding sites. In order to probe the assembly of the four calcium‐binding sites in S100b, a peptide comprised of the N‐terminal 46 residues of S100b protein was synthesized and studied by CD and 1H NMR spectroscopies as a function of concentration and temperature. At relatively high peptide concentrations and in the absence of calcium, the peptide exhibited a significant proportion of α‐helix (45%). Decreasing the peptide concentration led to a loss of α‐helix as monitored by CD spectroscopy and coincident changes in the 1H NMR spectrum. These changes were also observed by 1H NMR spectroscopy as a function of temperature where it was observed that the Tm of the peptide was lowered approximately 14 °C with a 17‐fold decrease in peptide concentration. Sedimentation equilibrium studies were used to determine that the peptide formed a tetramer in solution in the absence of calcium. It is proposed that this tetrameric fold also occurs in S100b and is a result of the interaction of portions of all four calcium‐binding sites.
Structural motifs for pyridoxal‐5′‐phosphate binding in decarboxylases: An analysis based on the crystal structure of the Lactobacillus 30a ornithine decarboxylase
Authors: Momany Cory , Ghosh Ratna , Hackert Marvin L. 
Published: June 2010

AbstractTwo of the five domains in the structure of the ornithine decarboxylase (OrnDC) from Lactobacillus 30a share similar structural folds around the pyridoxal‐5′‐phosphate (PLP)‐binding pocket with the aspartate aminotransferases (AspATs). Sequence comparisons focusing on conserved residues of the aligned structures reveal that this structural motif is also present in a number of other PLP‐dependent enzymes including the histidine, dopa, tryptophan, glutamate, and glycine decarboxylases as well as tryptophanase and serine‐hydroxymethyl transferase. However, this motif is not present in eukaryotic OrnDCs, the diaminopimelate decarboxylases, nor the Escherichia coli or oat arginine decarboxylases. The identification and comparison of residues involved in defining the different classes are discussed.
Backbone dynamics of the oligomerization domain of p53 determined from 15N NMR relaxation measurements
Authors: Clubb Robert T. , Omichinski James G. , Gronenborn Angela M. , Marius Clore G. , Sakaguchi Kazuyazu , Appella Ettore 
Published: June 2010

AbstractThe backbone dynamics of the tetrameric p53 oligomerization domain (residues 319–360) have been investigated by two‐dimensional inverse detected heteronuclear 1H‐15N NMR spectroscopy at 500 and 600 MHz. 15N T1, T2, and heteronuclear NOEs were measured for 39 of 40 non‐proline backbone NH vectors at both field strengths. The overall correlation time for the tetramer, calculated from the T1/T2 ratios, was found to be 14.8 ns at 35 °C. The correlation times and amplitudes of the internal motions were extracted from the relaxation data using the model‐free formalism (Lipari G, Szabo A, 1982, J Am Chem Soc 104:4546–4559). The internal dynamics of the structural core of the p53 oligomerization domain are uniform and fairly rigid, with residues 327–354 exhibiting an average generalized order parameter (S2) of 0.88 ± 0.08. The N‐ and C‐termini exhibit substantial mobility and are unstructured in the solution structure of p53. Residues located at the N‐ and C‐termini, in the β‐sheet, in the turn between the α‐helix and β‐sheet, and at the C‐terminal end of the α‐helix display two distinct internal motions that are faster than the overall correlation time. Fast internal motions (±20 ps) are within the extreme narrowing limit and are of uniform amplitude. The slower motions (0.6–2.2 ns) are outside the extreme narrowing limit and vary in amplitude. Four residues at the tetramer interface exhibit a small degree of conformational averaging as evidenced by 15N line broadening, possibly due to sliding or rolling of the helices at the interface of the two dimers that form the tetramer.
Predicting the helix packing of globular proteins by self‐correcting distance geometry
Authors: Mumenthaler C.H. , Braun W. 
Published: June 2010

AbstractA new self‐correcting distance geometry method for predicting the three‐dimensional structure of small globular proteins was assessed with a test set of 8 helical proteins. With the knowledge of the amino acid sequence and the helical segments, our completely automated method calculated the correct backbone topology of six proteins. The accuracy of the predicted structures ranged from 2.3 Å to 3.1 Å for the helical segments compared to the experimentally determined structures. For two proteins, the predicted constraints were not restrictive enough to yield a conclusive prediction. The method can be applied to all small globular proteins, provided the secondary structure is known from NMR analysis or can be predicted with high reliability.
Multimeric intermediates in the pathway to the aggregated inclusion body state for P22 tailspike polypeptide chains
Authors: Speed Margaret A. , Wang Daniel I.C. , King Jonathan 
Published: June 2010

AbstractThe failure of newly synthesized polypeptide chains to reach the native conformation due to their accumulation as inclusion bodies is a serious problem in biotechnology. The critical intermediate at the junction between the productive folding and the inclusion body pathway has been previously identified for the P22 tailspike endorham‐nosidase. We have been able to trap subsequent intermediates in the in vitro pathway to the aggregated inclusion body state. Nondenaturing gel electrophoresis identified a sequential series of multimeric intermediates in the aggregation pathway. These represent discrete species formed from noncovalent association of partially folded intermediates rather than aggregation of native‐like trimeric species. Monomer, dimer, trimer, tetramer, pentamer, and hexamer states of the partially folded species were populated in the initial stages of the aggregation reaction. This methodology of isolating early multimers along the aggregation pathway was applicable to other proteins, such as the P22 coat protein and carbonic anhydrase II.
Characterization of a quaternary‐structured folding intermediate of an antibody Fab‐fragment
Authors: Lilie Hauke , Jaenicke Rainer , Buchner Johannes 
Published: June 2010

AbstractAntibody folding is a complex process comprising folding and association reactions. Although it is usually difficult to characterize kinetic folding intermediates, in the case of the antibody Fab fragment, domain‐domain interactions lead to a rate‐limiting step of folding, thus accumulating folding intermediates at a late step of folding. Here, we analyzed a late folding intermediate of the Fab fragment of the monoclonal antibody MAK 33 from mouse (k/IgGl). As a strategy for accumulation of this intermediate we used partial denaturation of the native Fab by guanidinium chloride. This denaturation intermediate, which can be populated to about 90%, is indistinguishable from a late‐folding intermediate with respect to denaturation and renaturation kinetics. The spectroscopic analysis reveals a native‐like secondary structure of this intermediate with aromatic side chains only slightly more solvent exposed than in the native state. The respective partner domains are weakly associated. From these data we conclude that the intramolecular association of the two chains during folding, with all domains in a nativelike structure, follows a two‐step mechanism. In this mechanism, presumably hydrophobic interactions are followed by rearrangements leading to the exact complementarity of the contact sites of the respective domains.
Crystal structure of human pepsin and its complex with pepstatin
Authors: Fujinaga Masao , Chernaia Maia M. , Mosimann Steve C. , James Michael N.G. , Tarasova Nadya I. 
Published: June 2010

AbstractThe three‐dimensional crystal structure of human pepsin and that of its complex with pepstatin have been solved by X‐ray crystallographic methods. The native pepsin structure has been refined with data collected to 2.2 Å resolution to an R‐factor of 19.7%. The pepsin:pepstatin structure has been refined with data to 2.0 Å resolution to an R‐factor of 18.5%. The hydrogen bonding interactions and the conformation adopted by pepstatin are very similar to those found in complexes of pepstatin with other aspartic proteinases. The enzyme undergoes a conformational change upon inhibitor binding to enclose the inhibitor more tightly. The analysis of the binding sites indicates that they form an extended tube without distinct binding pockets. By comparing the residues on the binding surface with those of the other human aspartic proteinases, it has been possible to rationalize some of the experimental data concerning the different specificities. At the S1 site, valine at position 120 in renin instead of isoleucine, as in the other enzymes, allows for binding of larger hydrophobic residues. The possibility of multiple conformations for the P2 residue makes the analysis of the S2 site difficult. However, it is possible to see that the specific interactions that renin makes with histidine at P2 would not be possible in the case of the other enzymes. At the S3 site, the smaller volume that is accessible in pepsin compared to the other enzymes is consistent with its preference for smaller residues at the P3 position.
Crystallization and preliminary X‐ray diffraction studies on recombinant isopenicillin N synthase from Aspergillus nidulans
Authors: Roach Peter L. , Schofield Christopher J. , Baldwin Jack E. , Clifton Ian J. , Hajdu Janos 
Published: June 2010

AbstractRecombinant Aspergillus nidulans isopenicillin N synthase was purified from an Escherichia coli expression system. The apoenzyme in the presence of saturating concentrations of MnCl2 could be crystallized by either macro‐ or microseeding, using the hanging drop vapor diffusion technique with polyethylene glycol 8000 as precipitant. The crystals (0.5–1.0 mm overall dimensions) diffract X‐rays to at least 2.0 Å resolution at synchrotrons and belong to space group P212121 with unit cell dimensions of a = 59.2 Å, b = 127.0 Å, and c = 139.6 Å. The asymmetric unit contains one dimer, and the solvent content of the crystals is 60%. The crystals are radiation sensitive.
Crystallization of fragment D from human fibrinogen
Authors: Everse Stephen J. , Pelletier Huguette , Doolittle Russell F. 
Published: June 2010

AbstractFragment D from human fibrinogen has been crystallized. The fragment, which is composed of three disulfide‐linked chains (α' β' γ' = 88,000), was generated with either plasmin or mild trypsin digestion. The crystals diffracted out to 3.5 Å; the space group is P21, unit cell dimensions a = 108 Å, b = 48 Å, c = 167 Å, β = 106°. Fragment D was also co‐crystallized with the ligand GPRP‐amide, in which case the space group is consistent with P212121, unit cell dimensions a = 476 Å, b = 82 Å, c = 432 Å.
Interaction of SecB with intermediates along the folding pathway of maltose‐binding protein
Authors: Diamond Deborah L. , Strobel Sharon , Chun Sang‐Yearn , Randall Linda L. 
Published: June 2010

AbstractSecB, a molecular chaperone involved in protein export in Escherichia coli, displays the remarkable ability to selectively bind many different polypeptide ligands whose only common feature is that of being nonnative. The selectivity is explained in part by a kinetic partitioning between the folding of a polypeptide and its association with SecB. SecB has no affinity for native, stably folded polypeptides but interacts tightly with polypeptides that are nonnative. In order to better understand the nature of the binding, we have examined the interaction of SecB with intermediates along the folding pathway of maltose‐binding protein. Taking advantage of forms of maltose‐binding protein that are altered in their folding properties, we show that the first intermediate in folding, represented by the collapsed state, binds to SecB, and that the polypeptide remains active as a ligand until it crosses the final energy barrier to attain the native state.
On the role of Glu‐68 in alcohol dehydrogenase
Authors: Ryde Ulf 
Published: June 2010

AbstractTheoretical computations (molecular dynamics and combined quantum chemical and molecular mechanical geometry optimizations) have been performed on horse liver alcohol dehydrogenase. The results provide evidence that Glu‐68, a highly conserved residue located 0.47 nm from the catalytic zinc ion, may intermittently coordinate to the zinc ion. Structures with Glu‐68 coordinated to the zinc ion are almost as stable as structures with Glu‐68 at the crystal position and the barrier between the two configurations of Glu‐68 is so low that it can readily be bypassed at room temperature. There is a cavity behind the zinc ion that seems to be tailored to allow such coordination of Glu‐68 to the zinc ion. It is suggested that Glu‐68 may facilitate the exchange of ligands in the substrate site by coordinating to the zinc ion when the old ligand dissociates.
Comparison of methods for searching protein sequence databases
Authors: Pearson William R. 
Published: June 2010

AbstractWe have compared commonly used sequence comparison algorithms, scoring matrices, and gap penalties using a method that identifies statistically significant differences in performance. Search sensitivity with either the Smith‐Waterman algorithm or FASTA is significantly improved by using modern scoring matrices, such as BLOSUM45–55, and optimized gap penalties instead of the conventional PAM250 matrix. More dramatic improvement can be obtained by scaling similarity scores by the logarithm of the length of the library sequence (ln()‐scaling). With the best modern scoring matrix (BLOSUM55 or J093) and optimal gap penalties (‐12 for the first residue in the gap and —2 for additional residues), Smith‐Waterman and FASTA performed significantly better than BLASTP. With ln()‐scaling and optimal scoring matrices (BLOSUM45 or Gonnet92) and gap penalties (‐12, ‐1), the rigorous Smith‐Waterman algorithm performs better than either BLASTP and FASTA, although with the Gonnet92 matrix the difference with FASTA was not significant. Ln()‐scaling performed better than normalization based on other simple functions of library sequence length. Ln()‐scaling also performed better than scores based on normalized variance, but the differences were not statistically significant for the BLOSUM50 and Gonnet92 matrices. Optimal scoring matrices and gap penalties are reported for Smith‐Waterman and FASTA, using conventional or ln()‐scaled similarity scores. Searches with no penalty for gap extension, or no penalty for gap opening, or an infinite penalty for gaps performed significantly worse than the best methods. Differences in performance between FASTA and Smith‐Waterman were not significant when partial query sequences were used. However, the best performance with complete query sequences was obtained with the Smith‐Waterman algorithm and ln()‐scaling.
Automatic recognition of hydrophobic clusters and their correlation with protein folding units
Authors: Zehfus Micheal H. 
Published: June 2010

AbstractA method is described to objectively identify hydrophobic clusters in proteins of known structure. Clusters are found by examining a protein for compact groupings of side chains. Compact clusters contain seven or more residues, have an average of 65% hydrophobic residues, and usually occur in protein interiors. Although smaller clusters contain only side‐chain moieties, larger clusters enclose significant portions of the peptide backbone in regular secondary structure. These clusters agree well with hydrophobic regions assigned by more intuitive methods and many larger clusters correlate with protein domains. These results are in striking contrast with the clustering algorithm of J. Heringa and P. Argos (1991, J Mol Biol 220:151–171). That method finds that clusters located on a protein's surface are not especially hydrophobic and average only 3–4 residues in size.Hydrophobic clusters can be correlated with experimental evidence on early folding intermediates. This correlation is optimized when clusters with less than nine hydrophobic residues are removed from the data set. This suggests that hydrophobic clusters are important in the folding process only if they have enough hydrophobic residues.
Optimization of homoepitaxially grown AlGaN/GaN heterostructures
Authors: Grenko J. A. , Ebert C. W. , Reynolds C. L. , Duscher G. J. , Barlage D. W. , Johnson M. A. L. , Preble E. A. , Paskova T. , Evans K. R. 
Published: June 2010

AbstractWe report on the growth of Al0.25Ga0.75N/GaN heterostructures on low dislocation density semi‐insulating c‐axis GaN substrates by metalorganic vapor phase epitaxy (MOVPE). A room temperature (RT) Hall mobility (µRT) up to 2065 cm2 V−1 s−1 at sheet density (ns) of 8.25 × 1012 cm−2 has been measured. This work compliments prior studies in which we observed a buffer‐induced modulation of the RT two‐dimensional electron gas (2DEG) ns and µRT by varying the GaN buffer layer thickness. Here, we focus on the optimization of the heterostructure 2DEG properties by elimination of silicon doping in the Al0.25Ga0.75N barrier and unintentional Al in the not‐intentionally doped (n.i.d.) GaN buffer layer. The 15% improvement in µRT and ns relative to previous results is consistent with those predicted by Poisson solver calculations. Use of thick GaN buffers has minimized the theoretical mobility reduction based on intersubband scattering and has enabled us to determine the 2DEG sheet density associated with the polarization field ($n_{{\rm s}}^{{\rm polar}} $) to be ∼5 × 1012 cm−2.