bluuebunny/crossref_metadata_2025_split huggingface dataset:
1H, 13C, and 15N resonance assignments of Fusarium solani pisi cutinase and preliminary features of the structure in solution
Authors: Prompers Jeanine J. , Hilbers Cornelis W. , Groenewegen Anneke , Schaik René C. Van , Pepermans Henri A. M. 
Published: February 2009

AbstractEssentially complete (96%) sequence‐specific assignments were made for the backbone and side‐chain 1H, 13C, and 15N resonances of Fusarium solani pisi cutinase, produced as a 214‐residue heterologous protein in Escherichia coli, using heteronuclear NMR techniques. Three structural features were noticed during the assignment. (1) The secondary structure in solution corresponds mostly with the structure from X‐ray diffraction, suggesting that both structures are globally similar. (2) The HN of Ala32 has a strongly upfield‐shifted resonance at 3.97 ppm, indicative of an amide‐aromatic hydrogen bond to the indole ring of Trp69 that stabilizes the N‐terminal side of the parallel β‐sheet. (3) The NMR data suggest that the residues constituting the oxyanion hole are quite mobile in the free enzyme in solution, in contrast to the existence of a preformed oxyanion hole as observed in the crystal structure. Apparently, cutinase forms its oxyanion hole upon binding of the substrate like true lipases.
Domain organization of calbindin D28k as determined from the association of six synthetic EF‐hand fragments
Authors: Linse Sara , Thulin Eva , Gifford Lida K. , Radzewsky Dina , Hagan James , Åkerfeldt Karin S. , Wilk Rosemarie R. 
Published: February 2009

AbstractCalbindin D28k is an intracellular Ca2+‐binding protein containing six subdomains of EF‐hand type. The number and identity of the globular domains within this protein have been elucidated using six synthetic peptide fragments, each corresponding to one EF‐hand subdomain. All six peptides were mixed in equimolar amounts in the presence of 10 mM Ca2+ to allow for the reconstitution of domains. The mixture was compared to native calbindin D28k and to the sum of the properties of the individual peptides using circular dichroism (CD), fluorescence, and 1H NMR spectroscopy, as well as gel filtration and ion‐exchange chromatography. It was anticipated that if the peptides associate to form native‐like domains, the properties would be similar to those of the intact protein, whereas if they did not interact, they would be the same as the properties of the isolated peptides. The results show that the peptides in the mixture interact with one another. For example, the CD and fluorescence spectra for the mixture are very similar to those of the intact calbindin D28k suggesting that the mixed EF‐hand fragments associate to form a native‐like structure. To determine the number of domains and the subdomain composition of each domain in calbindin D28k, a variety of peptide combinations containing two to five EF‐hand fragments were studied. The spectral and chromatographic properties of all the mixtures containing less than six peptides were closer to the sum of the properties of the relevant individual peptides than to the mixture of the six peptides. The results strongly suggest that all six EF‐hands are packed into one globular domain. The association of the peptide fragments is observed to drive the folding of the individual subdomains. For example, one of the fragments, EF2, which is largely unstructured in isolation even in the presence of high concentrations of Ca2+, is considerably more structured in the presence of the other peptides, as judged by CD difference spectroscopy. The CD data also suggest that the packing between the individual subdomains is specific.
Covalent methionylation of escherichia coli methionyl‐tRNA synthethase: Identification of the labeled amino acid residues by matrix‐assisted laser desorption‐ionization mass spectrometry
Authors: Gillet Sylvie , Hountondji Codjo , Schmitter Jean‐Marie , Blanquet Sylvain 
Published: February 2009

AbstractMethionyl‐adenylate, the mixed carboxylic‐phosphoric acid anhydride synthesized by methionyl‐tRNA synthetase (MetRS) is capable of reacting with this synthetase or other proteins, by forming an isopeptide bond with the ϵ‐NH2 group of lysyl residues. It is proposed that the mechanism for the in vitro methionylation of MetRS might be accounted for by the in situ covalent reaction of methionyl‐adenylate with lysine side chains surrounding the active center of the enzyme, as well as by exchange of the label between donor and acceptor proteins. Following the incorporation of 7.0 ± 0.5 mol of methionine per mol of a monomeric truncated methionyl‐tRNA synthetase species, the enzymic activities of [32P]PPi‐ATP isotopic exchange and tRNAMet aminoacylation were lowered by 75% and more than 90%, respectively. The addition of tRNAMet protected the enzyme against inactivation and methionine incorporation. Matrix‐assisted laser desorption‐ionization mass spectrometry designated lysines‐114, ‐132, ‐142 (or ‐147), ‐270, ‐282, ‐335, ‐362, ‐402, ‐439, ‐465, and ‐547 of truncated methionyl‐tRNA synthetase as the target residues for covalent binding of methionine. These lysyl residues are distributed at the surface of the enzyme between three regions [114‐150], [270‐362], and [402‐465], all of which were previously shown to be involved in catalysis or to be located in the binding sites of the three substrates, methionine, ATP, and tRNA.
Direct sequence analysis of proteins by in‐source fragmentation during delayed ion extraction
Authors: Lennon John J. , Walsh Kenneth A. 
Published: February 2009

AbstractContinuous segments of amino acid sequence information as long as 41 residues have been deduced by interpretation of matrix‐assisted laser desorption/ionization‐generated ion signals dominated by cn fragmentation within the ion source of a linear time‐of‐flight mass spectrometer utilizing delayed ion extraction. The technique has been applied successively to five proteins of mass 12.2 kDa to 18.3 kDa, yielding segments of continuous sequence as long as 41 residues without the need for prior proteolytic fragmentation. Intact crosslinks such as disulfides or heme linkages interrupt the generation of these data.
Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding
Authors: Eppink Michel H. M. , Berkel Willem J. H. Van , Schreuder Herman A. 
Published: February 2009

AbstractA novel conserved sequence motif has been located among the flavoprotein hydroxylases. Based on the crystal structure and site‐directed mutagenesis studies of p‐hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, this amino acid fingerprint sequence is proposed to play a dual function in both FAD and NAD(P)H binding. In PHBH, the novel sequence motif (residues 153‐166) includes strand A4 and the N‐terminal part of helix H7. The conserved amino acids Asp 159, Gly 160, and Arg 166 are necessary for maintaining the structure. The backbone oxygen of Cys 158 and backbone nitrogens of Gly 160 and Phe 161 interact indirectly with the pyrophosphate moiety of FAD, whereas it is known from mutagenesis studies that the side chain of the moderately conserved His 162 is involved in NADPH binding.
Preliminary crystal structure determination of the alkaline protease from the Antarctic psychrophile pseudomonas aeruginosa
Authors: Villeret Vincent , Beeumen Jozef Van , Chessa Jean‐Pierre , Gerday Charles 
Published: February 2009

AbstractA cold alkaline protease, isolated from an Antarctic Pseudomonas aeruginosa strain, has been purified and crystallized. Large crystals were obtained in the presence of PEG 6000 at pH 7 and pH 8. They belong to the space group P212121. A complete data set to 2.1 Å resolution has been measured. The structure has been determined by the molecular replacement method using the coordinates of the mesophilic alkaline protease as a model. The molecular replacement solution displays a correlation coefficient of 0.39 and an R‐factor of 0.48. Subsequent inspection of the electron density map in the active site region has confirmed the correctness of the solution. Model building and structure refinement will be initiated when the protease sequence becomes fully available. This is the second report, following one on an a‐amylase, of the preliminary crystallographic characterization of a psychrophilic enzyme.
Purification, characterization, and crystallization of Escherichia coli ribokinase
Authors: Sigrell Jill A. , Cameron Alexander D. , Jones T. Alwyn , Mowbray Sherry L. 
Published: February 2009

AbstractRibokinase phosphorylates ribose to form ribose‐5‐phosphate in the presence of ATP and magnesium. The phosphorylated sugar can enter the pentose phosphate pathway or be used for the synthesis of nucleotides, histidine, and tryptophan. Ribokinase belongs to the PfkB family of carbohydrate kinases, for which no three‐dimensional structure is currently known. We describe an improved purification protocol for Escherichia coli ribokinase and give evidence from light‐scattering and gel filtration studies that the protein forms a dimer in solution. Several types of crystals are also described that have been obtained of apo ribokinase, ribokinase in the presence of ATP, and in a ternary complex with an ATP‐analogue and ribose. The latter crystals give the best X‐ray diffraction. A complete data set has been collected at the synchrotron source in Hamburg, to 2.6 Å resolution using a frozen crystal. The crystals belong to space group P6122 or P6522 with cell parameters a = b = 95 Å and c = 155 Å.
The partial charge of the nitrogen atom in peptide bonds
Authors: Milner‐White E. James 
Published: February 2009

AbstractA majority of the standard texts dealing with proteins portray the peptide link as a mixture of two resonance forms, in one of which the nitrogen atom has a positive charge. As a consequence, it is often believed that the nitrogen atom has a net positive charge. This is in apparent contradiction with the partial negative charge on the nitrogen that is used in force fields for molecular modeling. However, charges on resonance forms are best regarded as formal rather than actual charges and current evidence clearly favors a net negative charge for the nitrogen atom. In the course of the discussion, new ideas about the electronic structure of amides and the peptide bond are presented.
Identification of the binding site on S100B protein for the actin capping protein CapZ
Authors: Kilby Peter M. , Roberts GORDON C. K. , Van Eldik Linda J. 
Published: February 2009

AbstractThe calcium‐binding protein S100B binds to several potential target proteins, but there is no detailed information showing the location of the binding site for any target protein on S100B. We have made backbone assignments of the calcium‐bound form of S100B and used chemical‐shift changes in spectra of 15N‐labeled protein to locate the site that binds a peptide corresponding to residues 265‐276 from CapZα, the actin capping protein. The largest chemical‐shift changes are observed for resonances arising from residues around the C terminus of the C‐terminal helix of S100B and residues Val‐8 to Asp‐12 of the N‐terminal helix. These residues are close to but not identical to residues that have been identified by mutational analysis to be important in other S100 protein‐protein interactions. They make up a patch across the S100B dimer interface and include some residues that are quite buried in the structure of calcium‐free S100B. We believe we may have identified a binding site that could be common to many S100 protein—protein interactions.
De novo heme proteins from designed combinatorial libraries
Authors: Hecht Michael H. , Vogel Kathleen M. , Spiro Thomas G. , Rojas Nina R. L. , Kamtekar Satwik , Simons Cyrena T. , Mclean Jeremy E. , Farid Ramy S. 
Published: February 2009

AbstractWe previously reported the design of a library of de novo amino acid sequences targeted to fold into four‐helix bundles. The design of these sequences was based on a “binary code” strategy, in which the patterning of polar and non‐polar amino acids is specified explicitly, but the exact identities of the side chains is varied extensively (Kamtekar S, Schiffer JM, Xiong H, Babik JM, Hecht MH, 1993, Science 262:1680‐1685). Because of this variability, the resulting collection of amino acid sequences may include de novo proteins capable of binding biologically important cofactors. To probe for such binding, the de novo sequences were screened for their ability to bind the heme cofactor. Among an initial collection of 30 binary code sequences, 15 are shown to bind heme and form bright red complexes. Characterization of several of these de novo heme proteins demonstrated that their absorption spectra and resonance Raman spectra resemble those of natural cytochromes. Because the design of these sequences is based on global features of polar/nonpolar patterning, the finding that half of them bind heme highlights the power of the binary code strategy, and demonstrates that isolating de novo heme proteins does not require explicit design of the cofactor binding site. Because bound heme plays a key role in the functions of many natural proteins, these results suggest that binary code sequences may serve as initial prototypes for the development of large collections of functionally active de novo proteins.
Linkers of secondary structures in proteins
Authors: Geetha V. , Munson P. J. 
Published: February 2009

AbstractLinkers that connect repeating secondary structures fall into conformational classes based on distance and main‐chain torsion clustering. A data set of 300 unique protein chains with low pairwise sequence identity was clustered into only a few groups representing the preferred motifs. The linkers of two to eight residues for the nonredundant data set are designated H‐Ln‐H, H‐Ln‐E, E‐Ln‐H, E‐Ln‐E, where n is the length, H stands for α‐helices, and E for β‐strands. Most of the clusters identified here corroborate earlier findings. However, 19 new clusters are identified in this paper, with many of them having seven and eight residue linkers. In our first analysis, the secondary structures flanking the linkers are both interacting and noninteracting and there is no precise angle of orientation between them. A second analysis was performed on a set of proteins with restricted orientations for the flanking elements, namely, mainly α class of proteins with orthogonal architecture. Two definite clusters are identified, one corresponding to linkers of orthogonal helices and the other to linkers of antiparallel helices. Loops forming binding sites or involved in catalytic activity are important determinants of the function of proteins. Although the structural conservation of the residues around the catalytic triad of serine proteases has been studied widely, there has not been a systematic analysis of the conformation of the loops that contain them. Residues of the catalytic triad reside in the linkers of β‐strands, with varying lengths of more than eight residues. Here, we analyze the structural conservation of such linkers by superposition, and observe a conserved structural feature of the linkers incorporating each of the three residues of the catalytic triad.
Cross‐strand side‐chain interactions versus turn conformation in β‐hairpins
Authors: Alba Eva De , Rico Manuel , Jiménez M. Angeles 
Published: February 2009

AbstractA series of designed peptides has been analyzed by 1H‐NMR spectroscopy in order to investigate the influence of cross‐strand side‐chain interactions in β‐hairpin formation. The peptides differ in the N‐terminal residues of a previously designed linear decapeptide that folds in aqueous solution into two interconverting β‐hairpin conformations, one with a type I turn (β‐hairpin 4:4) and the other with a type I + G1 β‐bulge turn (β‐hairpin 3:5). Analysis of the conformational behavior of the peptides studied here demonstrates three favorable and two unfavorable cross‐strand side‐chain interactions for β‐hairpin formation. These results are in agreement with statistical data on side‐chain interactions in protein β‐sheets. All the peptides in this study form significant populations of the β‐hairpin 3:5, but only some of them also adopt the β‐hairpin 4:4. The formation of β‐hairpin 4:4 requires the presence of at least two favorable cross‐strand interactions, whereas β‐hairpin 3:5 seems to be less susceptible to side‐chain interactions. A protein database analysis of β‐hairpins 3:5 and β‐hairpins 4:4 indicates that the former occur more frequently than the latter. In both peptides and proteins, β‐hairpins 3:5 have a larger right‐handed twist than β‐hairpins 4:4, so that a factor contributing to the higher stability of β‐hairpin 3:5 relative to β‐hairpin 4:4 is due to an appropriate backbone conformation of the type I + G1 β‐bulge turn toward the right‐handed twist usually observed in protein β‐sheets. In contrast, as suggested previously, backbone geometry of the type I turn is not adequate for the right‐handed twist. Because analysis of buried hydrophobic surface areas on protein β‐hairpins reveals that β‐hairpins 3:5 bury more hydrophobic surface area than β‐hairpins 4:4, we suggest that the right‐handed twist observed in β‐hairpin 3:5 allows a better packing of side chains and that this may also contribute to its higher intrinsic stability.
Analysis of temperature factor distribution in high‐resolution protein structures
Authors: Parthasarathy S. , Murthy M.R.N. 
Published: February 2009

AbstractThe temperature factors obtained from X‐ray refinement of proteins at high resolution show large variations from one structure to another. However, the B‐values expressed in units of standard deviation about their mean value (B'‐factor) at the Cα atoms show remarkably characteristic frequency distribution. In all of the 110 proteins examined in this study, the frequency distribution exhibited a bimodal distribution. The peaks in the B'‐factor frequency distribution occur at ‐1.1 and 0.4 for a bin size of 0.5. The peak at lower temperature factor corresponds largely to buried residues, whereas the peak at larger value corresponds to exposed residues. The distribution could be accurately described as a superposition of two Gaussian functions. The parameters describing the distribution are therefore characteristic of protein structures. The frequency distribution for a given amino acid over all the proteins also shows a similar bimodal distribution, although the areas under the two Gaussians differ from one amino acid to another. The area under the frequency distribution curve for any interval in B'‐factor represents the propensity of the amino acid to occur in that interval. This propensity is related both to the hydrophilicity/hydrophobicity of the residue and the tendency of the residue to impose a different degree of rigidity on the polypeptide chain. The frequency distribution of stretches of high B'‐factors departs appreciably from that expected for a random distribution. The correlation in the B‐values of sequentially proximal residues is probably responsible for the bimodal distribution.
Simulating the minimum core for hydrophobic collapse in globular proteins
Authors: Tsai Jerry , Gerstein Mark , Levitt Michael 
Published: February 2009

AbstractTo investigate the nature of hydrophobic collapse considered to be the driving force in protein folding, we have simulated aqueous solutions of two model hydrophobic solutes, methane and isobutylene. Using a novel methodology for determining contacts, we can precisely follow hydrophobic aggregation as it proceeds through three stages: dispersed, transition, and collapsed. Theoretical modeling of the cluster formation observed by simulation indicates that this aggregation is cooperative and that the simulations favor the formation of a single cluster midway through the transition stage. This defines a minimum solute hydrophobic core volume. We compare this with protein hydrophobic core volumes determined from solved crystal structures. Our analysis shows that the solute core volume roughly estimates the minimum core size required for independent hydrophobic stabilization of a protein and defines a limiting concentration of nonpolar residues that can cause hydrophobic collapse. These results suggest that the physical forces driving aggregation of hydrophobic molecules in water is indeed responsible for protein folding.
Thermal unfolding of dodecameric glutamine synthetase: Inhibition of aggregation by urea
Authors: Nosworthy Neil J. , Ginsburg Ann 
Published: February 2009

AbstractThermal unfolding of dodecameric manganese glutamine synthetase (622,000 Mr) at pH 7 and ∼0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm ∼ 42°C; ΔH ≅ 0.9 J/g) followed by a large irreversible transition (Tm ∼ 81°C; ΔH≅ 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55°C) and inhibits aggregation accompanying unfolding at ≤50.2 mg protein/mL. With increasing temperature (30‐70°C) or incubation times at 25°C (5‐35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo‐first‐order (t1/2 = 1,030s at 20.0°C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax ∼ 64°C; ΔH = 17 ± 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (ΔH = 57 ± 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4:144‐1552), after correcting for the binding of urea to protein sites exposed during unfolding (‐42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.
Demonstration of protein—protein interaction specificity by NMR chemical shift mapping
Authors: Rajagopal Ponni , Waygood E. Bruce , Reizer Jonathan , Saier Milton H. , Klevit Rachel E. 
Published: February 2009

AbstractChemical shift mapping is becoming a popular method for studying protein‐protein interactions in solution. The technique is used to identify putative sites of interaction on a protein surface by detecting chemical shift perturbations in simple (1H, 15N )‐HSQC NMR spectra of a uniformly labeled protein as a function of added (unlabeled) target protein. The high concentrations required for these experiments raise questions concerning the possibility for non‐specific interactions being detected, thereby compromising the information obtained. We demonstrate here that the simple chemical shift mapping approach faithfully reproduces the known functional specificities among pairs of closely related proteins from the phosphoenolpyruvate:sugar phosphotransferase systems of Escherichia coli and Bacillus subtilis.