bluuebunny/crossref_metadata_2025_split huggingface dataset:
Penicillopepsin‐JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to kcat
Authors: Stubbs Marlene , Ngo Kenny Q.P. , Cunningham Annie , Pai Emil F. , Hofmann Theo , Tu Guang‐Chou , Cao Qing‐Na , Ward Michael 
Published: February 2009

AbstractThe nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin‐JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT‐pyrG1, was expressed in an aspartic proteinase‐free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin‐JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate‐enhancing effects of the presence of alanine residues in positions P3 and P2′ of substrates, are similar to those of penicillopepsin‐JT1, endothia‐pepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate‐enhancing effect was due to a hydrogen bond between the ‐NH‐of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C‐terminal to Asp215. Thr219 of penicillopepsin‐JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in kcat when a P3 residue was present in the substrate, which was similar to that of the wild‐type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the ‐NH‐ of P3 to the enzyme, the scissile peptide bond between P1 and P1′ becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.
Three‐dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors
Authors: Harel Michal , Kryger Gitay , Rosenberry Terrone L. , Mallender William D. , Lewis Terence , Fletcher Rodney J. , Guss J.Mitchell , Silman Israel , Sussman Joel L. 
Published: February 2009

AbstractWe have crystallized Drosophila melanogaster acetylcholinesterase and solved the structure of the native enzyme and of its complexes with two potent reversible inhibitors, 1,2,3,4‐tetrahydro‐N‐(phenylmethyl)‐9‐acridinamine and 1,2,3,4‐tetrahydro‐N‐(3‐iodophenyl‐methyl)‐9‐acridinamine—all three at 2.7 Å resolution. The refined structure of D. melanogaster acetylcholinesterase is similar to that of vertebrate acetylcholinesterases, for example, human, mouse, and fish, in its overall fold, charge distribution, and deep active‐site gorge, but some of the surface loops deviate by up to 8 Å from their position in the vertebrate structures, and the C‐terminal helix is shifted substantially. The active‐site gorge of the insect enzyme is significantly narrower than that of Torpedo californica AChE, and its trajectory is shifted several angstroms. The volume of the lower part of the gorge of the insect enzyme is ∼50% of that of the vertebrate enzyme. Upon binding of either of the two inhibitors, nine aromatic side chains within the active‐site gorge change their conformation so as to interact with the inhibitors. Some differences in activity and specificity between the insect and vertebrate enzymes can be explained by comparison of their three‐dimensional structures.
Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sUgar phosphotransferase system produced by substituting alanine or glutamate for the active‐site histidine 189: Implications for phosphorylation effects
Authors: Ginsburg Ann , Szczepanowski Roman H. , Ruvinov Sergei B. , Nosworthy Neil J. , Sondej Melissa , Umland Timothy C. , Peterkofsky Alan 
Published: February 2009

AbstractThe amino terminal domain of enzyme I (residues 1‐258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active‐site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature‐induced changes in ellipticity at 222 nm for monomeric wild‐type and mutant EIN proteins indicate two‐state unfolding. For EIN proteins in 10 mM K‐phosphate (and 100 mM KCl) at pH 7.5ΔH ≅ 140± 10 (160) kcal mol−1 and ΔCp ≅ 2.7 (3.3) kcal K−1 mol−1. Transition temperatures (Tm) are 57 (59), 55 (58), and 53 (56) °C for wild‐type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho‐His 189, phospho‐His189, and H189 substitutions of EIN at pH7.5 is: His > Ala > Glu > His‐PO32− due to differences in conformational entropy. Although H189E mutants have decreased Tm values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (Tm ∼ 66–68 °C). This possibly arises from an ion pair interaction between the γ‐carboxyl of Glu189 and the ϵ‐amino group of Lys69 in the docking region for the histidine‐containing phosphocarrier protein HPr. However, the binding of HPr to wild‐type and active‐site mutants of EIN and EI is temperature‐independent (entropically controlled) with about the same affinity constant at pH 7.5: K'A = 3 ± 1 × 105 M−1 for EIN and ∼1.2 × 105 M−1 for EI.
Conformational behavior of ionic self‐complementary peptides
Authors: Altman Michael , Lee Peter , Rich Alexander , Zhang Shuguang 
Published: February 2009

AbstractSeveral de novo designed ionic peptides that are able to undergo conformational change under the influence of temperature and pH were studied. These peptides have two distinct surfaces with regular repeats of alternating hydrophilic and hydrophobic side chains. This permits extensive ionic and hydrophobic interactions resulting in the formation of stable β‐sheet assemblies. The other defining characteristic of this type of peptide is a cluster of negatively charged aspartic or glutamic acid residues located toward the N‐terminus and positively charged arginine or lysine residues located toward the C‐terminus. This arrangement of charge balances the α‐helical dipole moment (C → N), resulting in a strong tendency to form stable α‐helices as well. Therefore, these peptides can form both stable α‐helices and β‐sheets. They are also able to undergo abrupt structural transformations between these structures induced by temperature and pH changes. The amino acid sequence of these peptides permits both stable β‐sheet and α‐helix formation, resulting in a balance between these two forms as governed by the environment. Some segments in proteins may also undergo conformational changes in response to environmental changes. Analyzing the plasticity and dynamics of this type of peptide may provide insight into amyloid formation. Since these peptides have dynamic secondary structure, they will serve to refine our general understanding of protein structure.
Prediction of amino acid sequence from structure
Authors: Raha Kaushik , Wollacott Andrew M. , Italia Michael J. , Desjarlais John R. 
Published: February 2009

AbstractWe have developed a method for the prediction of an amino acid sequence that is compatible with a three‐dimensional backbone structure. Using only a backbone structure of a protein as input, the algorithm is capable of designing sequences that closely resemble natural members of the protein family to which the template structure belongs. In general, the predicted sequences are shown to have multiple sequence profile scores that are dramatically higher than those of random sequences, and sometimes better than some of the natural sequences that make up the superfamily. As anticipated, highly conserved but poorly predicted residues are often those that contribute to the functional rather than structural properties of the protein. Overall, our analysis suggests that statistical profile scores of designed sequences are a novel and valuable figure of merit for assessing and improving protein design algorithms.
Tumor suppressor INK4: Refinement of p16INK4A structure and determination of p15INK4B structure by comparative modeling and NMR data
Authors: Yuan Chunhua , Selby Thomas L. , Li Junan , Byeon In‐Ja L. , Tsai Ming‐Daw 
Published: February 2009

AbstractWithin the tumor suppressor protein INK4 (inhibitor of cyclin‐dependent kinase 4) family, p15INK4B is the smallest and the only one whose structure has not been determined previously, probably due to the protein's conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16INK4A as the template, followed by restrained energy minimization with NMR constraints (NOE and H‐bonds). For this purpose, the solution structure of p16INK4A, whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure‐based iterative NOE assignments. The nonhelical regions showed major improvement with root‐mean‐square deviation (RMSD) improved from 1.23 to 0.68 Å for backbone heavy atoms. The completion of p15INK4B coupled with refinement of p16INK4A made it possible to compare the structures of the four INK4 members in depth, and to compare the structures of p16INK4A in the free form and in the p16INK4A‐CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each INK4 member.
Overall rotational diffusion and internal mobility in domain II of protein G from Streptococcus determined from 15N relaxation data
Authors: Tillett Marcus L. , Blackledge Martin J. , Derrick Jeremy P. , Lian Lu‐Yun , Norwood Timothy J. 
Published: February 2009

AbstractThe backbone dynamics and overall tumbling of protein G have been investigated using 15N relaxation. Comparison of measured R2/R1 relaxation rate ratios with known three‐dimensional coordinates of the protein show that the rotational diffusion tensor is significantly asymmetric, exhibiting a prolate axial symmetry. Extensive Monte Carlo simulations have been used to estimate the uncertainty due to experimental error in the relaxation rates to be D∥︁/D1 = 1.68± 0.08, while the dispersion in the NMR ensemble leads to a variation of Dn/D1 = 1.65 ± 0.03. Incorporation of this tensorial description into a Lipari‐Szabo type analysis of internal motion has allowed us to accurately describe the local dynamics of the molecule. This analysis differs from an earlier study where the overall rotational diffusion was described by a spherical top. In this previous analysis, exchange parameters were fitted to many of the residues in the alpha helix. This was interpreted as reflecting a small motion of the alpha helix with respect to the beta sheet. We propose that the differential relaxation properties of this helix compared to the beta sheet are due to the near‐orthogonality of the NH vectors in the two structural motifs with respect to the unique axis of the diffusion tensor. Our analysis shows that when anisotropic rotational diffusion is taken into account NH vectors in these structural motifs appear to be equally rigid. This study underlines the importance of a correct description of the rotational diffusion tensor if internal motion is to be accurately investigated.
Influence of the C‐terminus of the glycophorin A transmembrane fragment on the dimerization process
Authors: Orzáez Mar , Pérez‐Payá Enrique , Mingarro Ismael 
Published: February 2009

AbstractThe monomer–dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix–helix packing in a membrane‐mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C‐terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two‐stage model for membrane protein folding and oligomerization.
The active site and substrates binding mode of malonyl‐CoA synthetase determined by transferred nuclear Overhauser effect spectroscopy, site‐directed mutagenesis, and comparative modeling studies
Authors: Jung Jin‐Won , An Jae Hyung , Na Kyu Bong , Kim Yu Sam , Lee Weontae 
Published: February 2009

AbstractThe active sites and substrate bindings of Rhizobium trifolii molonyl‐CoA synthetase (MCS) catalyzing the malonyl‐CoA formation from malonate and CoA have been determined based on NMR spectroscopy, site‐directed mutagenesis, and comparative modeling methods. The MCS‐bound conformation of malonyl‐CoA was determined from two‐dimensional‐transferred nuclear Overhauser effect spectroscopy data. MCS protein folds into two structural domains and consists of 16 α‐helices, 24 β‐strands, and several long loops. The core active site was determined as a wide cleft close to the end of the small C‐terminal domain. The catalytic substrate malonate is placed between ATP and His206 in the MCS enzyme, supporting His206 in its catalytic role as it generates reaction intermediate, malonyl‐AMP. These findings are strongly supported by previous biochemical data, as well as by the site‐directed mutagenesis data reported here. This structure reveals the biochemical role as well as the substrate specificity that conservative residues of adenylate‐forming enzymes have.
The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry
Authors: Jamin Marc , Loh Stewart N. , Baldwin Robert L. , Antalik Marian , Bolen D. Wayne , Loh Stewart N. 
Published: February 2009

AbstractThe unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 °C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 °C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.
Autopalmitoylation of tubulin
Authors: Wolff J. , Zambito Anna Maria , Britto P. Jeram , Knipling Leslie 
Published: February 2009

AbstractPure rat brain tubulin is readily palmitoylated in vitro using [3H]palmitoyl CoA but no added enzymes. A maximum of approximately six palmitic acids are added per dimer in 2–3 h at 36–37 °C under native conditions. Both α and β tubulin are labeled, and 63–73% of the label was hydroxylamine‐labile, presumed thioesters. Labeling increases with increasing pH and temperature, and with low concentrations of guanidine HCl or KCl (but not with urea) to a maximum of ∼13 palmitates/dimer. High SDS and guanidine HCl concentrations are inhibitory. At no time could all 20 cysteine residues of the dimer be palmitoylated. Polymerization to microtubules, or use of tubulin S, markedly decreases the accessibility of the palmitoylation sites. Palmitoylation increases the electrophoretic mobility of a portion of α tubulin toward the β band. Palmitoylated tubulin binds a colchicine analogue normally, but during three warm/cold polymerization/ depolymerization cycles there is a progressive loss of palmitoylated tubulin, indicating decreased polymerization competence. We postulate that local electrostatic factors are major regulators of reactivity of tubulin cysteine residues toward palmitoyl CoA, and that the negative charges surrounding a number of the cysteines are sensitive to negative charges on palmitoyl CoA.
The X‐ray structure of the NAD‐dependent 5,10‐methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae
Authors: Monzingo Arthur F. , Breksa Andrew , Ernst Stephen , Appling Dean R. , Robertus Jon D. 
Published: February 2009

AbstractEucaryotes possess one or more NADP‐dependent methylene‐THF dehydrogenases as part of multifunctional enzymes. In addition, yeast expresses an unusual monofunctional NAD‐dependent enzyme, yMTD. We report X‐ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 Å resolution, respectively. The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes. The enzyme has two prominent domains, with the active site cleft between them. yMTD has a noncanonical NAD‐binding domain that has two inserted strands compared with the NADP‐binding domains of the bifunctional enzymes. This insert precludes yMTD from dimerizing in the same way as the bifunctional enzymes. yMTD functions as a dimer, but the mode of dimerization is novel. It does not appear that the difference in dimerization accounts for the difference in cofactor specificity or for the loss of cyclohydrolase activity. These functional differences are probably accounted for by minor differences within the tertiary structure of the active site of the monomeric protein.
HYR, an extracellular module involved in cellular adhesion and related to the immunoglobulin‐like fold
Authors: Callebaut Isabelle , Mornon Jean‐Paul , Gilgès Delphine , Vigon Isabelle 
Published: February 2009

AbstractDomains belonging to the immunoglobulin‐like fold are responsible for a wide variety of molecular recognition processes. Here we describe a new family of domains, the HYR family, which is predicted to belong to this fold, and which appears to be involved in cellular adhesion. HYR domains were identified in several eukaryotic proteins, often associated with Complement Control Protein (CCP) modules or arranged in multiple copies. Our analysis provides a sequence and structural basis for understanding the role of these domains in interaction mechanisms and leads to further characterization of heretofore undescribed repeated domains with similar folds found in several bacterial proteins involved in enzymatic activities (some chitinases) or in cell surface adhesion (streptococcal C‐alpha antigen).
Decoys ‘R’ Us: A database of incorrect conformations to improve protein structure prediction
Authors: Samudrala Ram , Levitt Michael 
Published: February 2009

AbstractThe development of an energy or scoring function for protein structure prediction is greatly enhanced by testing the func–tion on a set of computer–generated conformations (decoys) to determine whether it can readily distinguish native–like conforma–tions from nonnative ones. We have created “Decoys “R” Us,” a database containing many such sets of conformations, to provide a resource that allows scoring functions to be improved.
Structural effects of the active site mutation cysteine to serine in Bacillus cereus zinc–β–lactamase
Authors: Chantalat Laurent , Duée Emile , Dideberg Otto , Galleni Moreno , Frère Jean–Marie 
Published: February 2009

Abstractβ–Lactamases are involved in bacterial resistance. Mem–bers of the metallo–enzyme class are now found in many patho–genic bacteria and are becoming thus of major clinical importance. Despite the availability of Zn–β–lactamase X–ray structures their mechanism of action is still unclear. One puzzling observation is the presence of one or two zincs in the active site. To aid in assessing the role of zinc content in β–lactam hydrolysis, the re–placement by Ser of the zinc–liganding residue Cys168 in the Zn–β–lactamase from Bacillus cereus strain 569/H/9 was carried out: the mutant enzyme (C168S) is inactive in the mono–Zn form, but active in the di–Zn form. The structure of the mono–Zn form of the C168S mutant has been determined at 1.85 Å resolution. Ser168 occupies the same position as Cys168 in the wild–type enzyme. The protein residues mostly affected by the mutation are Asp90– Arg91 and His210. A critical factor for the activity of the mono–Zn species is the distance between Asp90 and the Zn ion, which is controlled by Arg91: a slight movement of Asp90 impairs cataly‐sis. The evolution of a large superfamily including Zn–β–lactamases suggests that they may not all share the same mechanism.
Folding of a three–stranded coiled coil
Authors: Dürr Eberhard , Bosshard Hans Rudolf 
Published: February 2009

AbstractCoiled coils consist of two or more amphipathic α–helices wrapped around each other to form a superhelical structure stabi–lized at the interhelical interface by hydrophobic residues spaced in a repeating 3–4 sequence pattern. Dimeric coiled coils have been shown to often form in a single step reaction in which asso–ciation and folding of peptide chains are tightly coupled. Here, we ask whether such a simple folding mechanism may also apply to the formation of a three–stranded coiled coil. The designed 29–residue peptide LZ16A was shown previously to be in a concen–tration–dependent equilibrium between unfolded monomer (M), folded dimer (D), and folded trimer (T). We show by time–resolved fluorescence change experiments that folding of LZ16A to D and T can be described by

The following rate constants were determined (25 °C, pH 7): k1 = 7.8×104M‐1s‐1, k‐1=0.015 s‐1, k2 = 6.5×105M‐1s−1, and k‐2 = 1.1 s‐1. In a separate experiment, equilibrium binding constants were determined from the change with concentration of the far–ultraviolet circular dichroism spectrum of LZ16A and were in good agreement with the kinetic rate constants according to KD = k1/2k‐1 and KT = k2/k‐2. Furthermore, pulsed hydrogen–exchange experiments indicated that only unfolded M and folded D and T were significantly populated during folding. The results are com–patible with a two–step reaction in which a subpopulation of as–sociation competent (e.g., partly helical) monomers associate to dimeric and trimeric coiled coils.