bluuebunny/crossref_metadata_2025_split huggingface dataset:
A new approach to the design of uniquely folded thermally stable proteins
Authors: Jiang Xin , Pistor Ernie , Farid Ramy S. , Farid Hany 
Published: October 2010

AbstractA new computer program (CORE) is described that predicts core hydrophobic sequences of predetermined target protein structures. A novel scoring function is employed, which for the first time incorporates parameters directly correlated to free energies of unfolding (DLGu), melting temperatures (Tm), and cooperativity. Metropolis‐driven simulated annealing and low‐temperature Monte Carlo sampling are used to optimize this score, generating sequences predicted to yield uniquely folded, stable proteins with cooperative unfolding transitions. The hydrophobic core residues of four natural proteins were predicted using CORE with the backbone structure and solvent exposed residues as input. In the two smaller proteins tested (Gβl, 11 core amino acids; 434 cro, 10 core amino acids), the native sequence was regenerated as well as the sequence of known thermally stable variants that exhibit cooperative denaturation transitions. Previously designed sequences of variants with lower thermal stability and weaker cooperativity were not predicted. In the two larger proteins tested (myoglobin, 32 core amino acids; methionine aminopeptidase, 63 core amino acids), sequences with corresponding side‐chain conformations remarkably similar to that of native were predicted.
Direct measurement of islet amyloid polypeptide fibrillogenesis by mass spectrometry
Authors: Larson Jennifer L. , Ko Eric , Miranker Andrew D. 
Published: October 2010

AbstractA novel method for monitoring fibrillogenesis is developed and applied to the amyloidogenic peptide, islet amyloid polypeptide (IAPP). The approach, based on electrospray ionization mass spectrometry, is complementary to existing assays of fibril formation as it monitors directly the population of precursor rather than product molecules. We are able to monitor fiber formation in two modes: a quenched mode in which fibril formation is halted by dilution into denaturant and a real time mode in which fibril formation is conducted within the capillary of the electrospray source. Central to the method is the observation that fibrillar IAPP does not compromise the ionization of monomeric IAPP. Furthermore, under mild ionization conditions, fibrillar IAPP does not dissociate and contribute to the monomeric signal. Critically, we introduce an internal standard, rat IAPP, for analysis on the mass spectrometer. This standard is sufficiently similar in sequence in that it ionizes identically to human IAPP. Furthermore, the sequence is sufficiently different in that it does not form fibrils and is distinguishable on the basis of mass. Applied to IAPP fibrillogenesis, our technique reveals that precursor consumption in seeded reactions obeys first‐order kinetics. Furthermore, a consistent level of monomer persists in both seeded and unseeded experiments after the fibril formation is complete. Given the inherent stability of fibrils, we expect this approach to be applicable to other amyloid systems.
A “structural” water molecule in the family of fatty acid binding proteins
Authors: Likić Vladimir A. , Prendergast Franklyn G. , Juranić Nenad , Macura Slobodan 
Published: October 2010

AbstractA single water molecule (w135), buried within the structure of rat intestinal fatty acid binding protein (I‐FABP), is investigated by NMR, molecular dynamics simulations, and analysis of known crystal structures. An ordered water molecule was found in structurally analogous position in 24 crystal structures of nine different members of the family of fatty acid binding proteins. There is a remarkable conservation of the local structure near the w135 binding site among different proteins from this family. NMR cross‐relaxation measurements imply that w135 is present in the I‐FABP:ANS (1‐sulfonato‐8‐(1′)anilinonaphthalene) complex in solution with the residence time of >300 ps. Mean‐square positional fluctuations of w135 oxygen observed in MD simulations (0.18 and 0.13 Å2) are comparable in magnitude to fluctuations exhibited by the backbone atoms and result from highly constrained binding pocket as revealed by Voronoi volumes (averages of 27.0 ± 1.8 Å3 and 24.7 ± 2.2 Å3 for the two simulations). Escape of w135 from its binding pocket was observed only in one MD simulation. The escape process was initiated by interactions with external water molecules and was accompanied by large deformations in β‐strands D and E. Immediately before the release, w135 assumed three distinct states that differ in hydrogen bonding topology and persisted for about 15 ps each. Computer simulations suggest that escape of w135 from the I‐FABP matrix is primarily determined by conformational fluctuations of the protein backbone and interactions with external water molecules.
The effect of nucleotide bias upon the composition and prediction of transmembrane helices
Authors: Stevens Timothy J. , Arkin Isaiah T. 
Published: October 2010

AbstractTransmembrane helices are the most readily predictable secondary structure components of proteins. They can be predicted to a high degree of accuracy in a variety of ways. Many of these methods compare new sequence data with the sequence characteristics of known transmembrane domains. However, the known transmembrane sequences are not necessarily representative of a particular organism. We attempt to demonstrate that parameters optimized for the known transmembrane domains are far from optimal when predicting transmembrane regions in a given genome. In particular, we have tested the effect of nucleotide bias upon the composition and hence the prediction characteristics of transmembrane helices. Our analysis shows that nucleotide bias of a genome has a strong and predictable influence upon the occurrences of several of the most important hydrophobic amino acids found within transmembrane helices. Thus, we show that nucleotide bias should be taken into account when determining putative transmembrane domains from sequence data.
Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium synechocystis sp. PCC 6803
Authors: Scott Nancy L. , Lecomte JULIETTE T. J. 
Published: October 2010

AbstractThe genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino‐acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme‐reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 μM. At neutral pH, it yielded an electronic absorption spectrum typical of a low‐spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low‐spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.
Mutational analysis of the major coat protein of M13 identifies residues that control protein display
Authors: Weiss Gregory A. , Wells James A. , Sidhu Sachdev S. 
Published: October 2010

AbstractWe have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487–495). Here, we explore how an optimized P8 variant (opti‐P8) could evolve the ability to efficiently display a protein fused to its N‐terminus. Reversion of individual opti‐P8 residues back to the wild‐type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein displayenhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities.
A method to predict residues conferring functional differences between related proteins: Application to MAP kinase pathways
Authors: Caffrey Daniel R. , Shields Denis C. , O'Neill LUKE A. J. 
Published: October 2010

AbstractPhysicochemical properties are potentially useful in predicting functional differences between aligned protein subfamilies. We present a method that considers physicochemical properties from ancestral sequences predicted to have given rise to the subfamilies of interest by gene duplication. Comparison between two map kinases subfamilies, p38 and ERK, revealed a region that had an excess of change in properties after gene duplication followed by conservation within the two subfamilies. This region corresponded to that experimentally defined as important for substrate and pathway specificity. The derived scores for the region of interest were found to differ significantly in their distribution compared to the rest of the protein when the Kolmogorov‐Smirnov test was applied (p = 0.005). Thus, the incorporation of ancestral physicochemical properties is useful in predicting functional differences between protein subfamilies. In addition, the method was applied to the MKK and MAPK components of the p38 and JNK pathways. These proteins showed a similar pattern in their evolution and regions predicted to confer functional differences are discussed.
Backbone dynamics measurements on leukemia inhibitory factor, a rigid four‐helical bundle cytokine
Authors: Yao Shenggen , Smith David K. , Hinds Mark G. , Zhang Jian‐Guo , Nicola Nicos A. , Norton Raymond S. 
Published: October 2010

AbstractThe backbone dynamics of the four‐helical bundle cytokine leukemia inhibitory factor (LIF) have been investigated using 15N NMR relaxation and amide proton exchange measurements on a murine—human chimera, MH35‐LIF. For rapid backbone motions (on a time scale of 10 ps to 100 ns), as probed by 15N relaxation measurements, the dynamics parameters were calculated using the model‐free formalism incorporating the model selection approach. The principal components of the inertia tensor of MH35‐LIF, as calculated from its NMR structure, were 1:0.98:0.38. The global rotational motion of the molecule was, therefore, assumed to be axially symmetric in the analysis of its relaxation data. This yielded a diffusion anisotropy D⟂D⟂ of 1.31 and an effective correlation time (4D⟂ + 2D⟂)−1 of 8.9 ns. The average values of the order parameters (S2) for the four helices, the long interhelical loops, and the N‐terminus were 0.91, 0.84, and 0.65, respectively, indicating that LIF is fairly rigid in solution, except at the N‐terminus. The S2 values for the long interhelical loops of MH35‐LIF were higher than those of their counterparts in short‐chain members of the four‐helical bundle cytokine family. Residues involved in LIF receptor binding showed no consistent pattern of backbone mobilities, with S2 values ranging from 0.71 to 0.95, but residues contributing to receptor binding site III had relatively lower S2 values, implying higher amplitude motions than for the backbone of sites I and II. In the relatively slow motion regime, backbone amide exchange measurements showed that a number of amides from the helical bundle exchanged extremely slowly, persisting for several months in 2H2O at 37 °C. Evidence for local unfolding was considered, and correlations among various structure‐related parameters and the backbone amide exchange rates were examined. Both sets of data concur in showing that LIF is one of the most rigid four‐helical bundle cytokines.
15N and 1H NMR study of histidine containing protein (hpr) from staphylococcus carnosus at high pressure
Authors: Kalbitzer Hans Robert , Görler Adrian , Li Hua , Dubovskii Peter V. , Hengstenberg Wolfgang , Kowolik Claudia , Yamada Hiroaki , Akasaka Kazuyuki 
Published: October 2010

AbstractThe pressure‐induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two‐dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side‐chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure‐induced conformational changes. The average pressure‐induced shifts for amide protons and nitrogens are 0.285 ppm GPa−1 at 278 K and 2.20 ppm GPa−1, respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa−1. Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the β‐pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure‐induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure‐induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four‐stranded β‐pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.
Crystal structure of adenosine kinase from Toxoplasma gondii at 1.8 Å resolution
Authors: Cook William J. , Delucas Lawrence J. , Chattopadhyay Debasish 
Published: October 2010

AbstractHuman infection with Toxoplasma gondii is an important cause of morbidity and mortality. Protozoan parasites such as T. gondii are incapable of de novo purine biosynthesis and must acquire purines from their host, so the purine salvage pathway offers a number of potential targets for antiparasitic chemotherapy. In T. gondii tachyzoites, adenosine is the predominantly salvaged purine nucleoside, and thus adenosine kinase is a key enzyme in the purine salvage pathway of this parasite. The structure of T. gondii adenosine kinase was solved using molecular replacement and refined by simulated annealing at 1.8 Å resolution to an R‐factor of 0.214. The overall structure and the active site geometry are similar to human adenosine kinase, although there are significant differences. The T. gondii adenosine kinase has several unique features compared to the human sequence, including a five‐residue deletion in one of the four linking segments between the two domains, which is probably responsible for a major change in the orientation of the two domains with respect to each other. These structural differences suggest the possibility of developing specific inhibitors of the parasitic enzyme.
Analysis of knowledge‐based protein‐ligand potentials using a self‐consistent method
Authors: Shimada J. , Ishchenko A.V. , Shakhnovich E.I. 
Published: October 2010

AbstractWe propose a self‐consistent approach to analyze knowledge‐based atom‐atom potentials used to calculate protein‐ligand binding energies. Ligands complexed to actual protein structures were first built using the SMoG growth procedure (DeWitte & Shakhnovich, 1996) with a chosen input potential. These model protein‐ligand complexes were used to construct databases from which knowledge‐based protein‐ligand potentials were derived. We then tested several different modifications to such potentials and evaluated their performance on their ability to reconstruct the input potential using the statistical information available from a database composed of model complexes. Our data indicate that the most significant improvement resulted from properly accounting for the following key issues when estimating the reference state: (1) the presence of significant nonenergetic effects that influence the contact frequencies and (2) the presence of correlations in contact patterns due to chemical structure. The most successful procedure was applied to derive an atom‐atom potential for real protein‐ligand complexes. Despite the simplicity of the model (pairwise contact potential with a single interaction distance), the derived binding free energies showed a statistically significant correlation (∼0.65) with experimental binding scores for a diverse set of complexes.
Structure of an analog of fusion peptide from hemagglutinin
Authors: Dubovskii Peter V. , Li Hua , Takahashi Sho , Arseniev Alexander S. , Akasaka Kazuyuki 
Published: October 2010

AbstractA 20‐residue peptide E5 containing five glutamates, an analog of the fusion peptide of influenza virus hemagglutinin (HA) exhibiting fusion activity at acidic pH lower than 6.0–6.5 was studied by circular dichroism (CD), Fourier transform infrared, and 1H‐NMR spectroscopy in water, water/trifluoroethanol (TFE) mixtures, dodecylphosphocholine (DPC) micelles, and phospholipid vesicles. E5 became structurally ordered at pH ≤6 and the helical content in the peptide increased in the row: water < water/tfe < dpc ∼ phospholipid vesicle while the amount of β‐structure was approximately reverse. 1H‐NMR data and line‐broadening effect of 5‐, 16‐doxylstearates on proton resonances of DPC bound peptide showed E5 forms amphiphilic α‐helix in residues 2–18, which is flexible in 11–18 part. The analysis of the proton chemical shifts of DPC bound and CD intensity at 220 nm of phospholipid bound E5 showed that the pH dependence of helical content is characterized by the same pKa ≈︁5.6. Only Glu11 and Glu15 in DPC bound peptide showed such elevated pKas, presumably due to transient hydrogen bond(s) Glu11 (Glu15) δCOO−(H+) … HN Glu15 that dispose(s) the side chain of Glu11 (Glu15) residue(s) close to the micelle/water interface. These glutamates are present in the HA‐fusion peptide and the experimental half‐maximal pH of fusion for HA and E5 peptides is ∼5.6. Therefore, a specific anchorage of these peptides onto membrane necessary for fusion is likely driven by the protonation of the carboxylate group of Glu11 (Glu 15) residue (s) participating in transient hydrogen bond(s).
Folding of a dimeric β‐barrel: Residual structure in the urea denatured state of the human papillomavirus E2 DNA binding domain
Authors: Alonso Leonardo G. , Prat‐Gay Gonzalo De , Bycroft Mark , Mok Yu‐Keung , Lima L. Mauricio T.R. 
Published: October 2010

AbstractThe dimeric β‐barrel is a characteristic topology initially found in the transcriptional regulatory domain of the E2 DNA binding domain from papillomaviruses. We have previously described the kinetic folding mechanism of the human HPV‐16 domain, and, as part of these studies, we present a structural characterization of the urea‐denatured state of the protein. We have obtained a set of chemical shift assignments for the C‐terminal domain in urea using heteronuclear NMR methods and found regions with persistent residual structure. Based on chemical shift deviations from random coil values, 3Jnhnα coupling constants, heteronuclear single quantum coherence peak intensities, and nuclear Overhauser effect data, we have determined clusters of residual structure in regions corresponding to the DNA binding helix and the second β‐strand in the folded conformation. Most of the structures found are of nonnative nature, including turn‐like conformations. Urea denaturation at equilibrium displayed a loss in protein concentration dependence, in absolute parallel to a similar deviation observed in the folding rate constant from kinetic experiments. These results strongly suggest an alternative folding pathway in which a dimeric intermediate is formed and the rate‐limiting step becomes first order at high protein concentrations. The structural elements found in the denatured state would collide to yield productive interactions, establishing an intermolecular folding nucleus at high protein concentrations. We discuss our results in terms of the folding mechanism of this particular topology in an attempt to contribute to a better understanding of the folding of dimers in general and intertwined dimeric proteins such as transcription factors in particular.
The crystal structures of glutathione S‐transferases isozymes 1–3 and 1–4 from Anopheles dirus species B
Authors: Oakley Aaron J. , Harnnoi Thasaneeya , Udomsinprasert Rungrutai , Jirajaroenrat Kanya , Ketterman Albert J. , Wilce Matthew C.J. 
Published: October 2010

AbstractGlutathione S‐transferases (GSTs) are dimeric proteins that play an important role in cellular detoxification. Four GSTs from the mosquito Anopheles dirus species B (Ad), an important malaria vector in South East Asia, are produced by alternate splicing of a single transcription product and were previously shown to have detoxifying activity towards pesticides such as DDT. We have determined the crystal structures for two of these alternatively spliced proteins, AdGST1–3 (complexed with glutathione) and AdGST1–4 (apo form), at 1.75 and 2.45 Å resolution, respectively. These GST isozymes show differences from the related GST from the Australian sheep blowfly Lucilia cuprina; in particular, the presence of a C‐terminal helix forming part of the active site. This helix causes the active site of the Anopheles GSTs to be enclosed. The glutathione‐binding helix α2 and flanking residues are disordered in the AdGST1–4 (apo) structure, yet ordered in the AdGST1–3 (GSH‐bound) structure, suggesting that insect GSTs operate with an induced fit mechanism similar to that found in the plant phi‐ and human pi‐class GSTs. Despite the high overall sequence identities, the active site residues of AdGST1–4 and AdGST1–3 have different conformations.