bluuebunny/crossref_metadata_2025_split huggingface dataset:
The oligomerization and ligand‐binding properties of Sm‐like archaeal proteins (SmAPs)
Authors: Mura Cameron , Kozhukhovsky Anna , Gingery Mari , Phillips Martin , Eisenberg David 
Published: March 2003

AbstractIntron splicing is a prime example of the many types of RNA processing catalyzed by small nuclear ribonucleoprotein (snRNP) complexes. Sm proteins form the cores of most snRNPs, and thus to learn principles of snRNP assembly we characterized the oligomerization and ligand‐binding properties of Sm‐like archaeal proteins (SmAPs) from Pyrobaculum aerophilum (Pae) and Methanobacterium thermautotrophicum (Mth). Ultracentrifugation shows that Mth SmAP1 is exclusively heptameric in solution, whereas Pae SmAP1 forms either disulfide‐bonded 14‐mers or sub‐heptameric states (depending on the redox potential). By electron microscopy, we show that Pae and Mth SmAP1 polymerize into bundles of well ordered fibers that probably form by head‐to‐tail stacking of heptamers. The crystallographic results reported here corroborate these findings by showing heptamers and 14‐mers of both Mth and Pae SmAP1 in four new crystal forms. The 1.9 Å‐resolution structure of Mth SmAP1 bound to uridine‐5′‐monophosphate (UMP) reveals conserved ligand‐binding sites. The likely RNA binding site in Mth agrees with that determined for Archaeoglobus fulgidus (Afu) SmAP1. Finally, we found that both Pae and Mth SmAP1 gel‐shift negatively supercoiled DNA. These results distinguish SmAPs from eukaryotic Sm proteins and suggest that SmAPs have a generic single‐stranded nucleic acid‐binding activity.
Analyses of circular dichroism spectra of membrane proteins
Authors: Wallace B.A. , Lees J.G. , Orry A.J.W. , Lobley A. , Janes Robert W. 
Published: March 2003

AbstractCircular dichroism (CD) spectroscopy is a valuable technique for the determination of protein secondary structures. Many linear and nonlinear algorithms have been developed for the empirical analysis of CD data, using reference databases derived from proteins of known structures. To date, the reference databases used by the various algorithms have all been derived from the spectra of soluble proteins. When applied to the analysis of soluble protein spectra, these methods generally produce calculated secondary structures that correspond well with crystallographic structures. In this study, however, it was shown that when applied to membrane protein spectra, the resulting calculations produce considerably poorer results. One source of this discrepancy may be the altered spectral peak positions (wavelength shifts) of membrane proteins due to the different dielectric of the membrane environment relative to that of water. These results have important consequences for studies that seek to use the existing soluble protein reference databases for the analyses of membrane proteins.
Anion binding to a protein–protein complex lacks dependence on net charge
Authors: Waldron Travis T. , Modestou Modestos A. , Murphy Kenneth P. 
Published: March 2003

AbstractThe binding of anions to proteins occurs in numerous physiological and metabolic processes. In an effort to understand the factors important in these interactions, we have studied the weak binding of phosphate and sulfate to a protein–protein complex using isothermal titration calorimetry. To our knowledge, this is the first system in which the thermodynamics of anion binding have been determined calorimetrically. By studying both phosphate and sulfate binding and using a range of pH values, the charge on the anion was varied from ∼ −1 to −2. Surprisingly, no dependence of the binding energetics on the charge of the anion was observed. This result indicates that charge–charge interactions are not the dominant factor in binding and suggests the importance of hydrogen bonding in specifically recognizing and coordinating anions.
Polycation‐induced oligomerization and accelerated fibrillation of human α‐synuclein in vitro
Authors: Goers John , Uversky Vladimir N. , Fink Anthony L. 
Published: March 2003

AbstractThe aggregation and fibrillation of α‐synuclein has been implicated as a causative factor in Parkinson's disease and several other neurodegenerative disorders known as synucleinopathies. The effect of different factors on the process of fibril formation has been intensively studied in vitro. We show here that α‐synuclein interacts with different unstructured polycations (spermine, polylysine, polyarginine, and polyethyleneimine) to form specific complexes. In addition, the polycations catalyze α‐synuclein oligomerization. The formation of α‐synuclein–polycation complexes was not accompanied by significant structural changes in α‐synuclein. However, α‐synuclein fibrillation was dramatically accelerated in the presence of polycations. The magnitude of the accelerating effect depended on the nature of the polymer, its length, and concentration. The results illustrate the potential critical role of electrostatic interactions in protein aggregation, and the potential role of naturally occurring polycations in modulating α‐synuclein aggregation.
NMR characterization of interleukin‐2 in complexes with the IL‐2Rα receptor component, and with low molecular weight compounds that inhibit the IL‐2/IL‐Rα interaction
Authors: Emerson S. Donald , Palermo Robert , Liu Chao‐Min , Tilley Jefferson W. , Chen Li , Danho Waleed , Madison Vincent S. , Greeley David N. , Ju Grace , Fry David C. 
Published: March 2003

AbstractNuclear magnetic resonance (NMR) methods were employed to study the interaction of the cytokine Interleukin‐2 (IL‐2) with the α‐subunit of its receptor (IL‐2Rα), and to help understand the behavior of small molecule inhibitors of this interaction. Heteronuclear 1H‐15N HSQC experiments were used to identify the interaction surface of 15N‐enriched Interleukin‐2 (15N‐IL‐2) in complex with human IL‐2Rα. In these experiments, chemical shift and line width changes in the heteronuclear single‐quantum coherence (HSQC) spectra upon binding of 15N‐IL‐2 enabled classification of NH atoms as either near to, or far from, the IL‐2Rα interaction surface. These data were complemented by hydrogen/deuterium (H/D) exchange measurements, which illustrated enhanced protection of slowly‐exchanging IL‐2 NH protons near the site of interaction with IL‐2Rα. The interaction surface defined by NMR compared well with the IL‐2Rα binding site identified previously using mutagenesis of human and murine IL‐2. Two low molecular weight inhibitors of the IL‐2/IL‐2Rα interaction were studied: one (a cyclic peptide derivative) was found to mimic a part of the cytokine and bind to IL‐2Rα; the other (an acylphenylalanine derivative) was found to bind to IL‐2. For the interaction between IL‐2 and the acylphenylalanine, chemical shift perturbations of 15N and 15NH backbone resonances were tracked as a function of ligand concentration. The perturbation pattern observed for this complex revealed that the acylphenylalanine is a competitive inhibitor—it binds to the same site on IL‐2 that interacts with IL‐2Rα.
Prediction of protein domain boundaries from sequence alone
Authors: Galzitskaya Oxana V. , Melnik Bogdan S. 
Published: March 2003

AbstractWe present here a simple approach to identify domain boundaries in proteins of an unknown three‐dimensional structure. Our method is based on the hypothesis that a high‐side chain entropy of a region in a protein chain must be compensated by a high‐residue interaction energy within the region, which could correlate with a well‐structured part of the globule, that is, with a domain unit. For protein domains, this means that the domain boundary is conditioned by amino acid residues with a small value of side chain entropy, which correlates with the side chain size. On the one hand, relatively high Ala and Gly content on the domain boundary results in high conformational entropy of the backbone chain between the domains. On the other hand, the presence of Pro residues leads to the formation of hinges for a relative orientation of domains. The method was applied to 646 proteins with two contiguous domains extracted from the SCOP database with a success rate of 63%. We also report the prediction of domain boundaries for CASP5 targets obtained with the same method.
The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient
Authors: Ho Jason G.S. , Middelberg Anton P.J. , Ramage Paul , Kocher Hans P. 
Published: March 2003

AbstractProtein aggregation is commonly observed during protein refolding. To better understand this phenomenon, the intermolecular interactions experienced by a protein during unfolding and refolding are inferred from second virial coefficient (SVC) measurements. It is accepted that a negative SVC is indicative of protein–protein interactions that are attractive, whereas a positive SVC indicates net repulsive interactions. Lysozyme denatured and reduced in guanidinium hydrochloride exhibited a decreasing SVC as the denaturant was diluted, and the SVC approached zero at approximately 3 M GdnHCl. Further dilution of denaturant to renaturation conditions (1.25 M GdnHCl) led to a negative SVC, and significant protein aggregation was observed. The inclusion of 500 mM L‐arginine in the renaturation buffer shifted the SVC to positive and suppressed aggregation, thereby increasing refolding yield. The formation of mixed disulfides in the denatured state prior to refolding also increased protein solubility and suppressed aggregation, even without the use of L‐arginine. Again, the suppression of aggregation was shown to be caused by a shift from attractive to repulsive intermolecular interactions as reflected in a shift from a negative to a positive SVC value. To the best of our knowledge, this is the first time that SVC data have been reported for renaturation studies. We believe this technique will aid in our understanding of how certain conditions promote renaturation and increase protein solubility, thereby suppressing aggregation. SVC measurements provide a useful link, for protein folding and aggregation, between empirical observation and thermodynamics.
Structure and dynamics of the DNA‐binding protein HU of B. stearothermophilus investigated by Raman and ultraviolet‐resonance Raman spectroscopy3
Authors: Serban Doinita , Arcineigas Sandra F. , Vorgias Constantinos E. , Thomas George J. 
Published: March 2003

AbstractThe histone‐like protein HU of Bacillus stearothermophilus (HUBst) is a 90‐residue homodimer that binds nonspecifically to B DNA. Although the structure of the HUBst:DNA complex is not known, the proposed DNA‐binding surface consists of extended arms that project from an α‐helical platform. Here, we report Raman and ultraviolet‐resonance Raman (UVRR) spectra diagnostic of subunit secondary structures and indicative of key side‐chains lining the proposed DNA‐binding surface. Raman conformation markers show that the DNA‐binding arms of the dimer contain β‐stranded structure in excess (eight ± two residues per subunit) of that reported previously. Important among side‐chain markers are Met (701 cm−1), Ala (908 cm−1), Arg (1082 cm−1), and Pro (1457 cm−1). The Ala marker undergoes a substantial shift (908 → 893 cm−1) on deuteration of alanyl peptide sites, indicating a coupled side‐chain/main‐chain mode of diagnostic value in the identification of exchange‐protected alanines. A large subset of alanines (67%) in the α‐helical core exhibits robust resistance to exchange. A quantitative study of NH → ND exchange exploiting newly identified amide II′ markers of helical (1440 cm−1) and nonhelical (1472 cm−1) conformations of HUBst indicates unexpected flexibility at the dimer interface, which is manifested in rapid exchange of 80% of peptide sites. The results establish a basis for subsequent Raman and UVRR investigations of HUBst:DNA complexes and provide a framework for applications to other DNA‐binding architectural proteins.
Tautomerism, acid‐base equilibria, and H‐bonding of the six histidines in subtilisin BPN′ by NMR
Authors: Day Regina M. , Thalhauser Craig J. , Sudmeier James L. , Vincent Matthew P. , Torchilin Ekaterina V. , Sanford David G. , Bachovchin Christopher W. , Bachovchin William W. 
Published: March 2003

AbstractWe have determined by 15N, 1H, and 13C NMR, the chemical behavior of the six histidines in subtilisin BPN′ and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every 15N, 1H, Cε1, and Cδ2 resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pKa = 7.30 ± 0.03 at 25°C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pKa value of 7.9 ± 0.3 and sluggish proton exchange behavior, as shown by two‐site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high Cε1‐H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved Cε1‐H…O=C H‐bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare Nδ1‐H tautomer, exhibiting 13Cδ1 chemical shifts ∼9 ppm higher than those for Nε2‐H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by 15N‐1H NOE effects, and titrates with rapid proton exchange kinetics linked to a pKa value of 7.47 ± 0.02.
Unexpected similarity in regulation between an archaeal inositol monophosphatase/fructose bisphosphatase and chloroplast fructose bisphosphatase
Authors: Stieglitz Kimberly A. , Seaton Barbara A. , Head James F. , Stec Boguslaw , Roberts Mary F. 
Published: March 2003

AbstractHyperthermophilic archaea have an unusual phosphatase that exhibits activity toward both inositol‐1‐phosphate and fructose‐1,6‐bisphosphate, activities carried out by separate gene products in eukaryotes and bacteria. The structures of phosphatases from Archaeoglobus fulgidus (AF2372) and Methanococcus jannaschii (MJ0109), both anaerobic organisms, resemble the dimeric unit of the tetrameric pig kidney fructose bisphosphatase (FBPase). A striking feature of AF2372, but not of MJ0109, is that the sulfhydryl groups of two cysteines, Cys150 and Cys186, are in close proximity (4 Å). A similar arrangement of cysteines has been observed in chloroplast FBPases that are regulated by disulfide formation controlled by redox signaling pathways (ferredoxin/thioredoxin). This mode of regulation has not been detected in any other FBPase enzymes. Biochemical assays show that the AF2372 phosphatase activity can be abolished by incubation with O2. Full activity is restored by incubation with thiol‐containing compounds. Neither the C150S variant of AF2372 nor the equivalent phosphatase from M. jannaschii loses activity with oxidation. Oxidation experiments using Escherichia coli thioredoxin, in analogy with the chloroplast FBPase system, indicate an unexpected mode of regulation for AF2372, a key phosphatase in this anaerobic sulfate reducer.
Rational design, synthesis, and verification of affinity ligands to a protein surface cleft
Authors: Baumann Herbert , Öhrman Sara , Shinohara Yasuro , Ersoy Oguz , Choudhury Devapriya , Axén Andreas , Tedebark Ulf , Carredano Enrique 
Published: March 2003

AbstractThe structure‐based design, synthesis, and screening of a glucuronic acid scaffold library of affinity ligands directed toward the catalytic cleft on porcine pancreas α‐amylase are presented. The design was based on the simulated docking to the enzyme active site of 53 aryl glycosides from the Available Chemicals Directory (ACD) selected by in silico screening. Twenty‐three compounds were selected for synthesis and screened in solution for binding toward α‐amylase using nuclear magnetic resonance techniques. The designed molecules include a handle outside of the binding site to allow their attachment to various surfaces with minimal loss of binding activity. After initial screening in solution, one affinity ligand was selected, immobilized to Sepharose (Amersham Biosciences), and evaluated as a chromatographic probe. A column packed with ligand‐coupled Sepharose specifically retained the enzyme, which could be eluted by a known inhibitor.
Prediction of the plant β‐barrel proteome: A case study of the chloroplast outer envelope
Authors: Schleiff Enrico , Eichacker Lutz Andreas , Eckart Kerstin , Becker Thomas , Mirus Oliver , Stahl Tanja , Soll Jürgen 
Published: March 2003

AbstractIn the postgenomic era, the transformation of genetic information into biochemical meaning is required. We have analyzed the proteome of the chloroplast outer envelope membrane by an in silico and a proteomic approach. Based on its evolutionary relation to the outer membrane of Gram‐negative bacteria, the outer envelope membrane should contain a large number of β‐barrel proteins. We therefore calculated the probability for the existence of β‐sheet, β‐barrel, and hairpin structures among all proteins of theArabidopsis thalianagenome. According to the existence of these structures, a number of candidates were selected. This protein pool was analyzed by TargetP to discard sequences with signals that would direct the protein to other organelles different from chloroplasts. In addition, the pool was manually controlled for the presence of proteins known to function outside of the chloroplast envelope. The approach developed here can be used to predict the topology of β‐barrel proteins. For the proteomic approach, proteins of highly purified outer envelope membranes of chloroplasts fromPisum sativumwere analyzed by ESI‐MS/MS mass spectrometry. In addition to the known components, four new proteins of the outer envelope membranes were identified in this study.
Tightly winding structure of sequential model peptide for repeated helical region in Samia cynthia ricini silk fibroin studied with solid‐state NMR
Authors: Nakazawa Yasumoto , Bamba Mie , Nishio Satoko , Asakura Tetsuo 
Published: March 2003

AbstractThere are many kinds of silks from silkworms and spiders with different structures and properties, and thus, silks are suitable to study the structure‐property relationship of fibrous proteins. Silk fibroin from a wild silkworm, Samia cynthia ricini, mainly consists of the repeated similar sequences by about 100 times where there are alternative appearances of the polyalanine (Ala)12–13 region and the Gly‐rich region. In this paper, a sequential model peptide, GGAGGGYGGDGG(A)12GGAGDGYGAG, which is a typical sequence of the silk fibroin, was synthesized, and the atomic‐level conformations of Gly residues at the N‐ and C‐terminal ends of the polyalanine region were determined as well as that of the central Ala residue using 13C 2D spin diffusion solid‐state nuclear magnetic resonance (NMR) under off‐magic angle spinning. In the model peptide with α‐helical conformation, the torsion angle of the central Ala residue, the 19th Ala, was determined to be (ϕ, ψ) = (−60°, −50°), which was a typical α‐helical structure, but the torsion angles of two Gly residues, the 12th and 25th Gly residues, which are located at the N‐ and C‐terminal ends of the polyalanine region, were determined to be (ϕ,ψ) = (−70°, −30°) and (ϕ,ψ) = (−70°, −20°), respectively. Thus, it was observed that the turns at both ends of polyalanine with α‐helix conformation in the model peptide are tightly wound.
Numerical Three-Dimensional Pressure Patterns in a Recess of a Turbulent and Compressible Hybrid Journal Bearing
Authors: Helene Mathieu , Arghir Mihai , Frene Jean 
Published: March 2003

The present work investigates the flow in the feeding recess of a hybrid journal bearing. Numerical integration of the complete Navier-Stokes equations was performed with an appropriate turbulence model. Of primary concern is the pressure field on the rotating journal surface that is commonly known as the recess pressure pattern. The goal of the work is to determine the influences of fluid compressibility, operating conditions and recess geometry. Reference parameters selected for this study comprise feeding Reynolds number Rea of 2.105, sliding Reynolds number Rec of 5.103 and recess depth over film thickness ratio e/H of 2.2. Compressibility was considered first. Three values of the axial exit Mach number were selected for computation, namely 0.2, 0.45, and 0.7. As no significant variation was found, the Mach number was fixed at 0.45 in subsequent studies concerning other parameters:     Feeding Reynolds number, Rea       2.104,2.105,4.105     Recess depth, e/H           0, 2.2, 8     Feedhole axis inclination        90°, 135°, 165°     Feedhole location (Figs. 1(a) and 13)   centered, downstream offset.
As each parameter is varied, wire mesh plot of pressure and its sectional profiles are examined and effects of varying various parameters are discussed in reference to flow processes as they may affect the support characteristics of the hybrid journal bearing.
MESOSCOPIC PATTERN FORMATION IN CATALYTIC PROCESSES BY AN EXTENSION OF THE MEAN FIELD APPROACH
Authors: MOLA E. E. , IRURZUN I. M. , VICENTE J. L. , KING D. A. 
Published: March 2003

For some years it has been known that a number of catalytic reactions, under specified steady operating conditions, exhibit oscillations, in the rate of product formation. These are often related to beautiful spatiotemporal patterns, including targets and spirals, on the metal surface. These examples of self-organizational phenomena have attracted considerable interest, because they are proving to be theoretically amenable. Here we review different approximations to model heterogeneous surface chemical reactions, which exhibit oscillatory behavior. A focal point is the use of a detailed knowledge of the dynamics of surface structural phase transition for modeling kinetic oscillations, which represent a severe test of our understanding of chemical processes at surfaces. Advantages and disadvantages of the Monte Carlo approach are presented to model heterogeneous oscillatory chemical reactions, with special emphasis if a Monte Carlo method is going to be applied to study the time evolution of a surface chemical reaction, as there should be a linear relationship between the time unit called the Monte Carlo step (MCS) and actual time. We conclude that special care must be taken when two or more processes are included in a simulation, because now overall MCS should be compatible with every individual process. The mean field approach (MFA) takes into account only reaction processes and completely neglects spatial correlation and fluctuations. Therefore, this approach is not adequate for describing the rich variety of spatial patterns that are experimentally observed. On the other hand, Monte Carlo approaches are severely limited by computational capabilities. To overcome MFA limitations we propose to extend the earlier work of King and coworkers [J. Chem. Phys.100, 14417 (1996)], which did not include spatial dependence, by adding diffusion processes and gas global coupling to the coupled reaction equations. The extended MFA can now be used as a new tool for the analysis of pattern formation in surface chemistry.