bluuebunny/crossref_metadata_2025_split huggingface dataset:
Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein
Authors: Shu Xiaokun , Wang Lei , Colip Leslie , Kallio Karen , Remington S. James 
Published: February 2009

AbstractmPlum is a far‐red fluorescent protein with emission maximum at ∼650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far‐red emission. Ultrafast time‐resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum‐E16Q and ‐I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far‐red emission. Both the far‐red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.
Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase
Authors: Conrath Katja , Pereira Alice S. , Martins Carlos E. , Timóteo Cristina G. , Tavares Pedro , Spinelli Silvia , Kinne Joerg , Flaudrops Christophe , Cambillau Christian , Muyldermans Serge , Moura Isabel , Moura Jose J. G. , Tegoni Mariella , Desmyter Aline 
Published: February 2009

AbstractNitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N2O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non‐hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies™). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.
Δ98Δ, a minimalist model of antiparallel β‐sheet proteins based on intestinal fatty acid binding protein
Authors: Curto Lucrecia María , Caramelo Julio Javier , Franchini Gisela Raquel , Delfino José María 
Published: February 2009

AbstractThe design of β‐barrels has always been a formidable challenge for de novo protein design. For instance, a persistent problem is posed by the intrinsic tendency to associate given by free edges. From the opposite standpoint provided by the redesign of natural motifs, we believe that the intestinal fatty acid binding protein (IFABP) framework allows room for intervention, giving rise to abridged forms from which lessons on β‐barrel architecture and stability could be learned. In this context, Δ98Δ (encompassing residues 29–126 of IFABP) emerges as a monomeric variant that folds properly, retaining functional activity, despite lacking extensive stretches involved in the closure of the β‐barrel. Spectroscopic probes (fluorescence and circular dichroism) support the existence of a form preserving the essential determinants of the parent structure, albeit endowed with enhanced flexibility. Chemical and physical perturbants reveal cooperative unfolding transitions, with evidence of significant population of intermediate species in equilibrium, structurally akin to those transiently observed in IFABP. The recognition by the natural ligand oleic acid exerts a mild stabilizing effect, being of a greater magnitude than that found for IFABP. In summary, Δ98Δ adopts a monomeric state with a compact core and a loose periphery, thus pointing to the nonintuitive notion that the integrity of the β‐barrel can indeed be compromised with no consequence on the ability to attain a native‐like and functional fold.
Fast structural dynamics in reduced and oxidized cytochrome c
Authors: Liu Weixia , Rumbley Jon N. , Englander S. Walter , Wand A. Joshua 
Published: February 2009

AbstractThe sub‐nanosecond structural dynamics of reduced and oxidized cytochrome c were characterized. Dynamic properties of the protein backbone measured by amide 15N relaxation and side chains measured by the deuterium relaxation of methyl groups change little upon change in the redox state. These results imply that the solvent reorganization energy associated with electron transfer is small, consistent with previous theoretical analyses. The relative rigidity of both redox states also implies that dynamic relief of destructive electron transfer pathway interference is not operational in free cytochrome c.
A protein encoded by a new family of mobile elements from Euryarchaea exhibits three domains with novel folds
Authors: Keller J. , Leulliot N. , Soler N. , Collinet B. , Vincentelli R. , Forterre P. , van Tilbeurgh H. 
Published: February 2009

AbstractWe present here the 2.6Å resolution crystal structure of the pT26‐6p protein, which is encoded by an ORF of the plasmid pT26‐2, recently isolated from the hyperthermophilic archaeon, Thermococcus sp. 26,2. This large protein is present in all members of a new family of mobile elements that, beside pT26‐2 include several virus‐like elements integrated in the genomes of several Thermococcales and Methanococcales (phylum Euryarchaeota). Phylogenetic analysis suggested that this protein, together with its nearest neighbor (organized as an operon) have coevolved for a long time with the cellular hosts of the encoding mobile element. As the sequences of the N and C‐terminal regions suggested a possible membrane association, a deletion construct (739 amino acids) was used for structural analysis. The structure consists of two very similar β‐sheet domains with a new topology and a five helical bundle C‐terminal domain. Each of these domains corresponds to a unique fold that has presently not been found in cellular proteins. This result supports the idea that proteins encoded by plasmid and viruses that have no cellular homologues could be a reservoir of new folds for structural genomic studies.
Crystal structure of AFV1‐102, a protein from the acidianus filamentous virus 1
Authors: Keller Jenny , Leulliot Nicolas , Collinet Bruno , Campanacci Valerie , Cambillau Christian , Pranghisvilli David , Tilbeurgh Herman van 
Published: February 2009

AbstractViruses infecting hyperthermophilic archaea have intriguing morphologies and genomic properties. The vast majority of their genes do not have homologs other than in other hyperthermophilic viruses, and the biology of these viruses is poorly understood. As part of a structural genomics project on the proteins of these viruses, we present here the structure of a 102 amino acid protein from acidianus filamentous virus 1 (AFV1‐102). The structure shows that it is made of two identical motifs that have poor sequence similarity. Although no function can be proposed from structural analysis, tight binding of the gateway tag peptide in a groove between the two motifs suggests AFV1‐102 is involved in protein protein interactions.
Structure and heme binding properties of Escherichia coli O157:H7 ChuX
Authors: Suits Michael D. L. , Lang Jérôme , Pal Gour P. , Couture Manon , Jia Zongchao 
Published: February 2009

AbstractFor many pathogenic microorganisms, iron acquisition from host heme sources stimulates growth, multiplication, ultimately enabling successful survival and colonization. In gram‐negative Escherichia coli O157:H7, Shigella dysenteriae and Yersinia enterocolitica the genes encoded within the heme utilization operon enable the effective uptake and utilization of heme as an iron source. While the complement of proteins responsible for heme internalization has been determined in these organisms, the fate of heme once it has reached the cytoplasm has only recently begun to be resolved. Here we report the first crystal structure of ChuX, a member of the conserved heme utilization operon from pathogenic E. coli O157:H7 determined at 2.05 Å resolution. ChuX forms a dimer which remarkably given low sequence homology, displays a very similar fold to the monomer structure of ChuS and HemS, two other heme utilization proteins. Absorption spectral analysis of heme reconstituted ChuX demonstrates that ChuX binds heme in a 1:1 manner implying that each ChuX homodimer has the potential to coordinate two heme molecules in contrast to ChuS and HemS where only one heme molecule is bound. Resonance Raman spectroscopy indicates that the heme of ferric ChuX is composed of a mixture of coordination states: 5‐coordinate and high‐spin, 6‐coordinate and low‐spin, and 6‐coordinate and high‐spin. In contrast, the reduced ferrous form displays mainly a 5‐coordinate and high‐spin state with a minor contribution from a 6‐coordinate and low‐spin state. The νFe‐CO and νC‐O frequencies of ChuX‐bound CO fall on the correlation line expected for histidine‐coordinated hemoproteins indicating that the fifth axial ligand of the ferrous heme is the imidazole ring of a histidine residue. Based on sequence and structural comparisons, we designed a number of site‐directed mutations in ChuX to probe the heme binding sites and dimer interface. Spectral analysis of ChuX and mutants suggests involvement of H65 and H98 in heme coordination as mutations of both residues were required to abolish the formation of the hexacoordination state of heme‐bound ChuX.
Relevance of weak flavin binding in human D‐amino acid oxidase
Authors: Caldinelli Laura , Molla Gianluca , Sacchi Silvia , Pilone Mirella S. , Pollegioni Loredano 
Published: February 2009

AbstractIn the brain, the human flavoprotein D‐amino acid oxidase (hDAAO) is involved in the degradation of the gliotransmitter D‐serine, an important modulator of NMDA‐receptor‐mediated neurotransmission; an increase in hDAAO activity (that yields a decrease in D‐serine concentration) was recently proposed to be among the molecular mechanisms leading to the onset of schizophrenia susceptibility. This human flavoenzyme is a stable homodimer (even in the apoprotein form) that distinguishes from known D‐amino acid oxidases because it shows the weakest interaction with the flavin cofactor in the free form. Instead, cofactor binding is significantly tighter in the presence of an active site ligand. In order to understand how hDAAO activity is modulated, we investigated the FAD binding process to the apoprotein moiety and compared the folding and stability properties of the holoenzyme and the apoprotein forms. The apoprotein of hDAAO can be distinguished from the holoenzyme form by the more “open” tertiary structure, higher protein fluorescence, larger exposure of hydrophobic surfaces, and higher sensitivity to proteolysis. Interestingly, the FAD binding only slightly increases the stability of hDAAO to denaturation by urea or temperature. Taken together, these results indicate that the weak cofactor binding is not related to protein (de)stabilization or oligomerization (as instead observed for the homologous enzyme from yeast) but rather should represent a means of modulating the activity of hDAAO. We propose that the absence in vivo of an active site ligand/substrate weakens the cofactor binding, yielding the inactive apoprotein form and thus avoiding excessive D‐serine degradation.
9,10‐Anthraquinone hinders β‐aggregation: How does a small molecule interfere with Aβ‐peptide amyloid fibrillation?
Authors: Convertino Marino , Pellarin Riccardo , Catto Marco , Carotti Angelo , Caflisch Amedeo 
Published: February 2009

AbstractAmyloid aggregation is linked to a number of neurodegenerative syndromes, the most prevalent one being Alzheimer's disease. In this pathology, the β‐amyloid peptides (Aβ) aggregate into oligomers, protofibrils, and fibrils and eventually into plaques, which constitute the characteristic hallmark of Alzheimer's disease. Several low‐molecular‐weight compounds able to impair the Aβ aggregation process have been recently discovered; yet, a detailed description of their interactions with oligomers and fibrils is hitherto missing. Here, molecular dynamics simulations are used to investigate the influence of two relatively similar tricyclic, planar compounds, that is, 9, 10‐anthraquinone (AQ) and anthracene (AC), on the early phase of the aggregation of the Aβ heptapeptide segment H14QKLVFF20, the hydrophobic stretch that promotes the Aβ self‐assembly. The simulations show that AQ interferes with β‐sheet formation more than AC. In particular, AQ intercalates into the β‐sheet because polar interactions between the compound and the peptide backbone destabilize the interstrand hydrogen bonds, thereby favoring disorder. The thioflavin T‐binding assay indicates that AQ, but not AC, sensibly reduces the amount of aggregated Aβ1–40 peptide. Taken together, the in silico and in vitro results provide evidence that structural perturbations by AQ can remarkably affect ordered oligomerization. Moreover, the simulations shed light at the atomic level on the interactions between AQ and Aβ oligomers, providing useful insights for the design of small‐molecule inhibitors of aggregation with therapeutic potential in Alzheimer's disease.
Building and assessing atomic models of proteins from structural templates: Learning and benchmarks
Authors: Vallat Brinda Kizhakke , Pillardy Jaroslaw , Májek Peter , Meller Jaroslaw , Blom Thomas , Cao BaoQiang , Elber Ron 
Published: February 2009

AbstractOne approach to predict a protein fold from a sequence (a target) is based on structures of related proteins that are used as templates. We present an algorithm that examines a set of candidates for templates, builds from each of the templates an atomically detailed model, and ranks the models. The algorithm performs a hierarchical selection of the best model using a diverse set of signals. After a quick and suboptimal screening of template candidates from the protein data bank, the current method fine‐tunes the selection to a few models. More detailed signals test the compatibility of the sequence and the proposed structures, and are merged to give a global fitness measure using linear programming. This algorithm is a component of the prediction server LOOPP (http://www.loopp.org). Large‐scale training and tests sets were designed and are presented. Recent results of the LOOPP server in CASP8 are discussed. Proteins 2009. © 2009 Wiley‐Liss, Inc.
Identification of protein functional surfaces by the concept of a split pocket
Authors: Tseng Yan Yuan , Li Wen‐Hsiung 
Published: February 2009

AbstractThe function of a protein is often fulfilled via molecular interactions on its surfaces, so identifying the functional surface(s) of a protein is helpful for understanding its function. Here, we introduce the concept of a split pocket, which is a pocket that is split by a cognate ligand. We use a geometric approach that is site‐specific. Specifically, we first compute a set of all pockets in the protein with its ligand(s) and a set of all pockets with the ligand(s) removed and then compare the two sets of pockets to identify the split pocket(s) of the protein. To reduce the search space and expedite the process of surface partitioning, we design probe radii according to the physicochemical textures of molecules. Our method achieves a success rate of 96% on a benchmark test set. We conduct a large‐scale computation to identify ∼19,000 split pockets from 11,328 structures (1.16 million potential pockets); for each pocket, we obtain residue composition, solvent‐accessible area, and molecular volume. With this database of split pockets, our method can be used to predict the functional surfaces of unbound structures. Indeed, the functional surface of an unbound protein may often be found from its similarity to remotely related bound forms that belong to distinct folds. Finally, we apply our method to identify glucose‐binding proteins, including unbound structures. Our study demonstrates the power of geometric and evolutionary matching for studying protein functional evolution and provides a framework for classifying protein functions by local spatial patterns of functional surfaces. Proteins 2009. © 2009 Wiley‐Liss, Inc.
Dynamics of β3 integrin I‐like and hybrid domains: Insight from simulations on the mechanism of transition between open and closed forms
Authors: Gaillard Thomas , Dejaegere Annick , Stote Roland H. 
Published: February 2009

AbstractThe conformational dynamics of the I‐like and Hybrid domains from the β3 integrin headpiece were studied by molecular dynamics simulation and normal mode analysis. Crystallographic structures of integrins show that the integrin headpiece can exist in largely different conformations manifested by a significant difference in the angle between the I‐like and Hybrid domains. The relative orientation of these two domains is believed to be a crucial element of integrin function, as it may relate local structural modifications induced by ligand binding into large‐scale conformational changes. To investigate the detailed mechanisms responsible for this coupling, we carried out molecular dynamics simulations of the I‐like/Hybrid system and employed quasi‐harmonic and normal mode analyses to characterize the large‐scale motions. Our results show that the conformational transition of I‐like and Hybrid domains inferred from crystallographic data is contained in the low‐frequency dynamics of the system. Using targeted molecular dynamics simulations, we investigated the roles played by two structural elements of the I‐like domain, the α7 and α1 helices, in the interdomain transition. From our results, we propose that these two helices function in tandem to initiate large‐scale, interdomain conformational transition apparent in integrin activation and signaling. Proteins 2009. © 2009 Wiley‐Liss, Inc.
Observations on the current status of Orobanche and Striga problems worldwide
Authors: Parker Chris 
Published: February 2009

AbstractSpecies of Orobanche and Striga are among the most damaging parasitic weed species worldwide, but there are few reliable statistics on the full extent of the economic losses they cause. The distribution, host range and economic importance of the major species of Orobanche and Striga are briefly summarised. A review of literature over the period since 1991 suggests that many million hectares are infested and that the losses amount to $ US billions annually. Unfortunately there are almost no fully reliable figures on which to base these figures precisely. Meanwhile, there is little evidence of any significant change in intensity, range or losses caused over this period. Any reduction in the importance of these damaging weeds is sporadic, and alleviation of the problems is mostly localised. Furthermore, while the importance of Orobanche species may be broadly static, Striga species on cereals continue to become more serious in many countries owing to continued loss of soil fertility. It is suggested that new techniques may be needed for measurement of the extent of losses caused by these genera and their economic impact. There is continued urgency to develop control measures appropriate to the farming systems involved, and to reduce the risk of spread of both groups of parasite to new areas. Copyright © 2009 Society of Chemical Industry
Laser microdissection and its application to analyze gene expression in arbuscular mycorrhizal symbiosis
Authors: Gomez S Karen , Harrison Maria J 
Published: February 2009

AbstractPhosphorus is essential for plant growth, and in many soils phosphorus availability limits crop production. Most plants in natural ecosystems obtain phosphorus via a symbiotic partnership with arbuscular mycorrhizal (AM) fungi. While the significance of these associations is apparent, their molecular basis is poorly understood. Consequently, the potential to harness the mycorrhizal symbiosis to improve phosphorus nutrition in agriculture is not realized. Transcript profiling has recently been used to investigate gene expression changes that accompany development of the AM symbiosis. While these approaches have enabled the identification of AM‐symbiosis‐associated genes, they have generally involved the use of RNA from whole mycorrhizal roots. Laser microdissection techniques allow the dissection and capture of individual cells from a tissue. RNA can then be isolated from these samples and cell‐type specific gene expression information can be obtained. This technology has been applied to obtain cells from plants and more recently to study plant–microbe interactions. The latter techniques, particularly those developed for root–microbe interactions, are of relevance to plant‐parasitic weed research. Here, laser microdissection, its use in plant biology and in particular plant–microbe interactions are discussed. An overview of the AM symbiosis is then provided, with a focus on recent advances in understanding development of the arbuscule–cortical cell interface. Finally, the recent applications of laser microdissection for analyses of AM symbiosis are discussed. Copyright © 2009 Society of Chemical Industry
Synthetic pentapeptides inhibiting autophosphorylation of insulin receptor in a non‐ATP‐competitive mechanism
Authors: Kato Masaki , Abe Mineo , Kuroda Yoshihiro , Hirose Munetaka , Nakano Minoru , Handa Tetsurou 
Published: February 2009

AbstractIn an attempt to develop non‐ATP‐competitive inhibitors of the autophosphorylation of IR, the effects of the synthetic peptides, Ac‐DIY1158ET‐NH2 and Ac‐DY1162Y1163RK‐NH2, on the phosphorylation of IR were studied in vitro. The peptides were derived from the amino‐acid sequence in the activation loop of IR. They inhibited the autophosphorylation of IR to 20.5 and 40.7%, respectively, at 4000 µM. The Asp/Asn‐ and Glu/Gln‐substituted peptides, Ac‐NIYQT‐NH2 and Ac‐NYYRK‐NH2, more potently inhibited the autophosphorylation than did the corresponding parent peptides. The inhibitory potencies of the substituted peptides were decreased with increasing concentrations of ATP, indicating that these peptides employ an ATP‐competitive mechanism in inhibiting the autophosphorylation of IR. In contrast, those of the parent peptides were not affected. Mass spectrometry showed that the parent peptides were phosphorylated by IR, suggesting that they interact with the catalytic loop. Moreover, docking simulations predicted that the substituted peptides would interact with the ATP‐binding region of IR, whereas their parent peptides would interact with the catalytic loop of IR. Thus, Ac‐DIYET‐NH2 and Ac‐DYYRK‐NH2 are expected to be non‐ATP‐competitive inhibitors. These peptides could contribute to the development of a drug employing a novel mechanism. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.