bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessing the role of aromatic residues in the amyloid aggregation of human muscle acylphosphatase
Authors: Bemporad Francesco , Taddei Niccolò , Stefani Massimo , Chiti Fabrizio 
Published: April 2006

AbstractAmong the many parameters that have been proposed to promote amyloid fibril formation is the π‐stacking of aromatic residues. We have studied the amyloid aggregation of several mutants of human muscle acylphosphatase in which an aromatic residue was substituted with a non‐aromatic one. The aggregation rate was determined using the Thioflavin T test under conditions in which the variants populated initially an ensemble of partially unfolded conformations. Substitutions in aggregation‐promoting fragments of the sequence result in a dramatically decreased aggregation rate of the protein, confirming the propensity of aromatic residues to promote this process. Nevertheless, a statistical analysis shows that the measured decrease of aggregation rate following mutation arises predominantly from a reduction of hydrophobicity and intrinsic β‐sheet propensity. This suggests that aromatic residues favor aggregation because of these factors rather than for their aromaticity.
Src kinase activation: A switched electrostatic network
Authors: Ozkirimli Elif , Post Carol Beth 
Published: April 2006

AbstractSrc tyrosine kinases are essential in numerous cell signaling pathways, and improper functioning of these enzymes has been implicated in many diseases. The activity of Src kinases is regulated by conformational activation, which involves several structural changes within the catalytic domain (CD): the orientation of two lobes of CD; rearrangement of the activation loop (A‐loop); and movement of an α‐helix (αC), which is located at the interface between the two lobes, into or away from the catalytic cleft. Conformational activation was investigated using biased molecular dynamics to explore the transition pathway between the active and the down‐regulated conformation of CD for the Src‐kinase family member Lyn kinase, and to gain insight into the interdependence of these changes. Lobe opening is observed to be a facile motion, whereas movement of the A‐loop motion is more complex requiring secondary structure changes as well as communication with αC. A key result is that the conformational transition involves a switch in an electrostatic network of six polar residues between the active and the down‐regulated conformations. The exchange between interactions links the three main motions of the CD. Kinetic experiments that would demonstrate the contribution of the switched electrostatic network to the enzyme mechanism are proposed. Possible implications for regulation conferred by interdomain interactions are also discussed.
Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study
Authors: Rajan Rahul S. , Li Tiansheng , Aras Mohini , Sloey Christopher , Sutherland Weston , Arai Hiromi , Briddell Robert , Kinstler Olaf , Lueras Alexis M.K. , Zhang Yu , Yeghnazar Heather , Treuheit Michael , Brems David N. 
Published: April 2006

AbstractPolyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N‐terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG‐GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG‐GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG‐GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG‐GCSF and 20kPEG‐GCSF. It was observed that even 5kPEG‐GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG‐GCSF. Biological activity measurements demonstrated that both 5kPEG‐GCSF and 20kPEG‐GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.
Sequential reorganization of β‐sheet topology by insertion of a single strand
Authors: Sagermann Martin , Baase Walter A. , Matthews Brian W. 
Published: April 2006

AbstractInsertions, duplications, and deletions of sequence segments are thought to be major evolutionary mechanisms that increase the structural and functional diversity of proteins. Alternative splicing, for example, is an intracellular editing mechanism that is thought to generate isoforms for 30%–50% of all human genes. Whereas the inserted sequences usually display only minor structural rearrangements at the insertion site, recent observations indicate that they may also cause more dramatic structural displacements of adjacent structures. In the present study we test how artificially inserted sequences change the structure of the β‐sheet region in T4 lysozyme. Copies of two different β‐strands were inserted into two different loops of the β‐sheet, and the structures were determined. Not surprisingly, one insert “loops out” at its insertion site and forms a new small β‐hairpin structure. Unexpectedly, however, the second insertion leads to displacement of adjacent strands and a sequential reorganization of the β‐sheet topology. Even though the insertions were performed at two different sites, looping out occurred at the C‐terminal end of the same β‐strand. Reasons as to why a non‐native sequence would be recruited to replace that which occurs in the native protein are discussed. Our results illustrate how sequence insertions can facilitate protein evolution through both local and nonlocal changes in structure.
Structure and dynamics of the epidermal growth factor receptor C‐terminal phosphorylation domain
Authors: Lee Nam Y. , Hazlett Theodore L. , Koland John G. 
Published: April 2006

AbstractThe C‐terminal phosphorylation domain of the epidermal growth factor receptor is believed to regulate protein kinase activity as well as mediate the assembly of signal transduction complexes. The structure and dynamics of this proposed autoregulatory domain were examined by labeling the extreme C terminus of the EGFR intracellular domain (ICD) with an extrinsic fluorophore. Fluorescence anisotropy decay analysis of the nonphosphorylated EGFR‐ICD yielded two rotational correlation times: a longer time, consistent with the global rotational motion of a 60‐ to 70‐kDa protein with an elongated globular conformation, and a shorter time, presumably contributed by segmental motion near the fluorophore. A C‐terminally truncated form of EGFR‐ICD yielded a slow component consistent with the rotational motion of the 38‐kDa kinase core. These findings suggested a structural arrangement of the EGFR‐ICD in which the C‐terminal phosphorylation domain interacts with the kinase core to move as an extended structure. A marked reduction in the larger correlation time of EGFR‐ICD was observed upon its autophosphorylation. This dynamic component was faster than predicted for the globular motion of the 62‐kDa EGFR‐ICD, suggesting an increase in the mobility of the C‐terminal domain and a likely displacement of this domain from the kinase core. The interaction between the SH2 domain of c‐Src and the phosphorylated EGFR C‐terminal domain was shown to impede its mobility. Circular dichroism spectroscopy indicated that the EGFR C‐terminal domain possessed a significant level of secondary structure in the form of α‐helices and β‐sheets, with a marginal change in β‐sheet content occurring upon phosphorylation.
Calorimetric and structural studies of the nitric oxide carrier S‐nitrosoglutathione bound to human glutathione transferase P1‐1
Authors: Téllez‐Sanz Ramiro , Cesareo Eleonora , Nuccetelli Marzia , Aguilera Ana M. , Barón Carmen , Parker Lorien J. , Adams Julian J. , Morton Craig J. , Lo Bello Mario , Parker Michael W. , García‐Fuentes Luis 
Published: April 2006

AbstractThe nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S‐nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1‐1 (hGSTP1‐1) at 1.4 Å resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild‐type hGSTP1‐1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild‐type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S‐nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix α2 of hGST P1‐1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild‐type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix α2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S‐nitrosylation of Cys47 by GSNO is a likely physiological scenario.
Modular organization of FDH: Exploring the basis of hydrolase catalysis
Authors: Reuland Steven N. , Vlasov Alexander P. , Krupenko Sergey A. 
Published: April 2006

AbstractAn abundant enzyme of liver cytosol, 10‐formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase‐homologous domain and a folate‐binding hydrolase domain, which are connected by an ∼100‐residue linker. The amino‐terminal hydrolase domain of FDH (Nt‐FDH) is a homolog of formyl transferase enzymes that utilize 10‐formyl‐THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+‐dependent oxidation of 10‐formyltetrahydrofolate (10‐formyl‐THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl‐tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt‐FDH and related 10‐formyl‐THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10‐formyl‐THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis.
The crystal structure of YycH involved in the regulation of the essential YycFG two‐component system in Bacillus subtilis reveals a novel tertiary structure
Authors: Szurmant Hendrik , Zhao Haiyan , Mohan Michael A. , Hoch James A. , Varughese Kottayil I. 
Published: April 2006

AbstractThe Bacillus subtilis YycFG two‐component signal transduction system is essential for cell viability, and the YycH protein is part of the regulatory circuit that controls its activity. The crystal structure of YycH was solved by two‐wavelength selenium anomalous dispersion data, and was refined using 2.3 Å data to an R‐factor of 25.2%. The molecule is made up of three domains, and has a novel three‐dimensional structure. The N‐terminal domain features a calcium binding site and the central domain contains two conserved loop regions.
Enhancing functional production of G protein‐coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen
Authors: André Nicolas , Cherouati Nadia , Prual Cécile , Steffan Tania , Zeder‐Lutz Gabrielle , Magnin Thierry , Pattus Franc , Michel Hartmut , Wagner Renaud , Reinhart Christoph 
Published: April 2006

AbstractWe have optimized the expression level of 20 mammalian G protein‐coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding‐competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.
Crystal structure of the conserved protein TTHA0727 from Thermus thermophilus HB8 at 1.9 Å resolution: A CMD family member distinct from carboxymuconolactone decarboxylase (CMD) and AhpD
Authors: Ito Kaori , Arai Ryoichi , Fusatomi Emiko , Kamo‐Uchikubo Tomomi , Kawaguchi Shin‐ichi , Akasaka Ryogo , Terada Takaho , Kuramitsu Seiki , Shirouzu Mikako , Yokoyama Shigeyuki 
Published: April 2006

AbstractTTHA0727 is a conserved hypothetical protein from Thermus thermophilus HB8, with a molecular mass of 12.6 kDa. TTHA0727 belongs to the carboxymuconolactone decarboxylase (CMD) family (Pfam 02627). A sequence comparison with its homologs suggested that TTHA0727 is a distinct protein from alkylhydroperoxidase AhpD and γ‐carboxymuconolactone decarboxylase in the CMD family. Here we report the 1.9 Å crystal structure of TTHA0727 (PDB ID: 2CWQ) determined by the multiwavelength anomalous dispersion method. The TTHA0727 monomer structure consists of seven α‐helices (α1–α7) and one short 310‐helix. The crystal structure and the analytical ultracentrifugation revealed that TTHA0727 forms a hexameric ring structure in solution. The electrostatic potential distribution on the solvent‐accessible surface of the TTHA0727 hexamer showed that positively charged regions exist on the side of the ring structure, suggesting that TTHA0727 interacts with some negatively charged molecules. A structural homology search revealed that the structure of three α‐helices (α4–α6) is remarkably conserved, suggesting that it is the common structural motif for the CMD family proteins. In addition, the nine residues of the N‐terminal tag bound to the cleft region between α1 and α3 in chains A and B of TTHA0727, implying that this region is the putative binding/active site for some small molecules.
THE PROTOPLASMIC ULTRASTRUCTURE OF THE VEGETATIVE CELL OF MELOSIRA VARIANS C. A. AGARDH1
Authors: Crawford Richard M. 
Published: April 2006

SUMMARYA detailed fine structural study of the nondividing vegetative cell of the centric diatom Melosira varians reveals peripheral organization of cytoplasm and organelles around a central vacuole. Polarity of the cell is such that the nucleus is found at the valve face of the epitheca. The unusual shape of the nucleus and its associated microtubular system and polar body are thought to relate to the migration of the nucleus during other phases of cell growth. Unusual discontinuities of the plastid lamellae are reported and the significance of the periplastid reticulum is discussed. Possible roles for the cytoplasmic vesicles and the membrane system in the cell are also considered.
ULTRASTRUCTURE OF SPERMATIUM LIBERATION IN THE MARINE RED ALGA PTILOTA DENSA1
Authors: Scott J. L. , Dixon P. S. 
Published: April 2006

SUMMARYThe differentiation of male gametes of the marine red alga Ptilota densa was studied by electron microscopy. Mature primary spermatangia are enveloped by a single cell wall and possess a clearly polar subcellular organization. The nucleus is situated apical to large, striated, fibrous vacuoles which are apparently formed by the repeated fusion of dictyosome vesicles. The transformation and liberation of spermatia from spermatangia involve both the secretion of the fibrous vacuoles at the base of the cell and the subsequent rupturing of the spermatangial cell wall. Liberated spermatia are coated with a thin mucilage layer and contain numerous small vesicles and several mitochondria and dictyosomes. The nucleus is cup‐shaped and generally lacks a limiting envelope. These findings are discussed in relation to other light and electron microscopic studies of differentiating spermatangia in red algae.
PERIDINIUM BALTICUM (LEVANDER) LEMMERMANN, AN UNUSUAL DINOFLAGELLATE WITH A MESOCARYOTIC AND AN EUCARYOTIC NUCLEUS123
Authors: Tomas Ronald N. , Cox Elenor R. , Steidinger Karen A. 
Published: April 2006

SUMMARYLight and electron microscopy indicate that Peridinium balticum possesses 2 Feulgen‐positive, membrane‐bound nuclei which divide synchronously. One nucleus has the typical structure of dinocaryotic dinoflagellates, while the other nucleus has a structure typical of eucaryotic organisms. Connections between each nucleus and the endoplasmic reticulum are common. Membrane‐bound vesicles are intimately associated with the nuclear envelope of the eucaryotic nucleus.
AGGREGATION OF SOIL PARTICLES BY ALGAE1
Authors: Bailey Don , Mazurak Andrew P. , Rosowski James R. 
Published: April 2006

SUMMARY Chlorella pyrenoidosa Chick (Indiana University Number 252), Nostoc commune Vaucher (I.U. 584), and Oscillatoria prolifica (Grev.) Gomont (I.U. 1270) were grown separately on Peoria loess soil material to measure their effects on the water stability of soil aggregates. Each alga significantly (10% LSD) increased the percentage of soil aggregates after 6 weeks of incubation as compared with the soil without algae. Oscillatoria, Chlorella, and Nostoc increased water stability of aggregates >74 μ in diameter by 3.4, 1.1, and 0.6%, respectively. Nostoc and Oscillatoria produced measurable water stable aggregates in the 1000–2000 μ diameter range; Chlorella formed them in the 500–1000 μ range, while the control soil showed no aggregates >295 μ.
EFFECT OF INCUBATION TEMPERATURE ON THE MACROMOLECULAR AND LIPID CONTENT OF THE PHYTOFLAGELLATE OCHROMONAS DANICA1
Authors: Aaronson S. 
Published: April 2006

SUMMARY Ochromonas danica grown in a chemically defined medium under controlled conditions at different incubation temperatures showed marked changes in chemical composition and secretory pattern. As the incubation temperature rose from 15 to 30 C, the cell number per unit volume of medium increased; the cell mass reached a maximum and then declined slightly, and the weight per single cell decreased. The chemical composition of the cells changed with increasing temperature: carbohydrates decreased dramatically; proteins quadrupled at 20 C and then declined 25%; lipids increased; and RNA increased to 25 C and then decreased to about 40% of maximum at 30 C. The extracellular secretion by the cells of carbohydrates seemed to increase and then decline; RNA decreased; proteins increased; and lipids remained approximately constant, as the temperature increased. The implications of these findings for thermal pollution are discussed.
PROTOTHECA: AN ALGA OR A FUNGUS?1
Authors: Nadakavukaren Mathew J. , McCracken Derek A. 
Published: April 2006

SUMMARYAlthough Prototheca is generally considered to be a colorless member of the Chlorococcales, there have been a number of attempts to relate it to the fungi. However, our ultrastructural observations support the conclusion that it is indeed a nonphotosynthetic alga and not a fungus. One such structural feature that is of special interest is the presence of membrane‐bounded starch granules which are considered to be storage plastids. Fungi are not known to produce plastids.
Induction of systemic resistance in tomato by N‐acyl‐L‐homoserine lactone‐producing rhizosphere bacteria
Authors: SCHUHEGGER REGINA , IHRING ALEXANDRA , GANTNER STEPHAN , BAHNWEG GüNTHER , KNAPPE CLAUDIA , VOGG GERD , HUTZLER PETER , SCHMID MICHAEL , VAN BREUSEGEM FRANK , EBERL LEO , HARTMANN ANTON , LANGEBARTELS CHRISTIAN 
Published: April 2006

ABSTRACT N‐acyl‐L‐homoserine lactone (AHL) signal molecules are utilized by Gram‐negative bacteria to monitor their population density (quorum sensing) and to regulate gene expression in a density‐dependent manner. We show that Serratia liquefaciens MG1 and Pseudomonas putida IsoF colonize tomato roots, produce AHL in the rhizosphere and increase systemic resistance of tomato plants against the fungal leaf pathogen, Alternaria alternata. The AHL‐negative mutant S. liquefaciens MG44 was less effective in reducing symptoms and A. alternata growth as compared to the wild type. Salicylic acid (SA) levels were increased in leaves when AHL‐producing bacteria colonized the rhizosphere. No effects were observed when isogenic AHL‐negative mutant derivatives were used in these experiments. Furthermore, macroarray and Northern blot analysis revealed that AHL molecules systemically induce SA‐ and ethylene‐dependent defence genes (i.e. PR1a, 26 kDa acidic and 30 kDa basic chitinase). Together, these data support the view that AHL molecules play a role in the biocontrol activity of rhizobacteria through the induction of systemic resistance to pathogens.