bluuebunny/crossref_metadata_2025 huggingface dataset:
Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct
Authors: Chen Guo‐Qiang , Sun Yu , Jin Rongsheng , Gouaux Eric
Published: February 2009

Ionotropic glutamate receptors constitute an important family of ligand‐gated ion channels for which there is little biochemical or structural data. Here we probe the domain structure and boundaries of the ligand binding domain of the AMPA‐sensitive GluR2 receptor by limited proteolysis and deletion mutagenesis. To identify the proteolytic fragments, Maldi mass spectrometry and N‐terminal amino acid sequencing were employed. Trypsin digestion of HS1S2 (Chen GQ, Gouaux E. 1997.Proc Natl Acad Sci USA 94:13431–13436) in the presence and absence of glutamate showed that the ligand stabilized the S1 and S2 fragments against complete digestion. Using limited proteolysis and multiple sequence alignments of glutamate receptors as guides, nine constructs were made, folded, and screened for ligand binding activity. From this screen, the S1S2I construct proved to be trypsin‐ and chymotrypsin‐resistant, stable to storage at 4 °C, and amenable to three‐dimensional crystal formation. The HS1S2I variant was readily prepared on a large scale, the His tag was easily removed by trypsin, and crystals were produced that diffracted to beyond 1.5 Å resolution. These experiments, for the first time, pave the way to economical overproduction of the ligand binding domains of glutamate receptors and more accurately map the boundaries of the ligand binding domain.
Effect of denaturant and protein concentrations upon protein refolding and aggregation: A simple lattice model
Authors: Gupta Prashant , Hall Carol K. , Voegler Anne C.
Published: February 2009

We present a study of the competition between protein refolding and aggregation for simple lattice model proteins. The effect of solvent conditions (i.e., the denaturant concentration and the protein concentration) on the folding and aggregation behavior of a system of simple, two‐dimensional lattice protein molecules has been investigated via dynamic Monte Carlo simulations. The population profiles and aggregation propensities of the nine most populated intermediate configurations exhibit a complex dependence on the solution conditions that can be understood by considering the competition between intra‐ and interchain interactions. Some of these configurations are not even seen in isolated chain simulations; they are observed to be highly aggregation prone and are stabilized primarily by the aggregation reaction in multiple‐chain systems. Aggregation arises from the association of partially folded intermediates rather than from the association of denatured random‐coil states. The aggregation reaction dominates over the folding reaction at high protein concentration and low denaturant concentration, resulting in low refolding yields at those conditions. However, optimum folding conditions exist at which the refolding yield is a maximum, in agreement with some experimental observations.
Selective and asymmetric action of trypsin on the dimeric forms of seminal RNase
Authors: Lorenzo Claudia De , Piccoli Renata , Maro Antimo Di , Pucci Piero , D'alessio Giuseppe , Piaz Fabrizio Dal
Published: February 2009

Dimeric seminal RNase (BS‐RNase) is an equilibrium mixture of conformationally different quaternary structures, one characterized by the interchange between subunits of their N‐terminal ends (the MXM form); the other with no interchange (the M=M form). Controlled tryptic digestion of each isolated quaternary form generates, as limit digest products, folded and enzymatically active molecules, very resistant to further tryptic degradation. Electrospray mass spectrometric analyses and N‐terminal sequence determinations indicate that trypsin can discriminate between the conformationally different quaternary structures of seminal RNase, and exerts a differential and asymmetric action on the two dimeric forms, depending on the original quaternary conformation of each form. The two digestion products from the MXM and the M=M dimeric forms have different structures, which are reminiscent of the original quaternary conformation of the dimers: one with interchange, the other with no interchange, of the N‐terminal ends. The surprising resistance of these tryptic products to further tryptic action is explained by the persistence in each digestion product of the original intersubunit interface.
Solution structure of the His 12 → Cys mutant of the N‐terminal zinc binding domain of HIV‐1 integrase complexed to cadmium
Authors: Cai Mengli , Caffrey Michael , Clore G. Marius , Gronenborn Angela M. , Huang Ying , Zheng Ronglan , Craigie Robert
Published: February 2009

The solution structure of His 12 → Cys mutant of the N‐terminal zinc binding domain (residues 1–55; IN1–55) of HIV‐1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild‐type N‐terminal domain complexed to zinc. In contrast to the wild‐type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His 12 → Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10–18 is intermediate between the two forms of the wild‐type complex.
Ca2+‐dependent conformational changes in bovine GCAP‐2
Authors: Hughes Robert E. , Hurley James B. , Brzovic Peter S. , Dizhoor Alexander M. , Klevit Rachel E.
Published: February 2009

GCAP‐2, a mammalian photoreceptor‐specific protein, is a Ca2+‐dependent regulator of the retinal membrane guanylyl cyclases (Ret‐GCs). Sensing the fall in intracellular free Ca2+ after photo‐excitation, GCAP‐2 stimulates the activity of Ret‐GC leading to cGMP production. Like other members of the recoverin superfamily, GCAP‐2 is a small N‐myristoylated protein containing four EF‐hand consensus motifs. In this study, we demonstrate that like recoverin and neurocalcin, GCAP‐2 alters its conformation in response to Ca2+‐binding as measured by a Ca2+‐dependent change in its far UV CD spectrum. Differences in the conformation of the Ca2+‐bound and Ca2+‐free forms of GCAP‐2 were also observed by examining their relative susceptibility to V8 protease. In contrast to recoverin, we do not observe proteolytic cleavage of the myristoylated N‐terminus of Ca2+‐bound GCAP‐2. NMR spectra also show that, in contrast to recoverin, the chemical environment of the N‐terminus of GCAP‐2 is not dramatically altered by Ca2+ binding. Despite the similarity of GCAP‐2 and recoverin, the structural consequences of Ca2+‐binding for these two proteins are significantly dissimilar.
A solution SAXS study of borrelia burgdorferi OspA, a protein containing a single‐layer β‐sheet
Authors: Bu Zimei , Engelman Donald M. , Koide Shohei
Published: February 2009

The crystal structure of a soluble form of Borrelia burgdorferi outer surface protein A (OspA) complexed with the Fab fragment of a monoclonal antibody has revealed an unusual structure that has a repetitive antiparallel β topology with a nonglobular, single layer β‐sheet connecting the globular N‐ and C‐terminal domains. Earlier NMR studies have shown that the local structure of OspA including the single layer β‐sheet is similar to the crystal structure. Here we report a small angle X‐ray scattering (SAXS) study of the global conformation of OspA in solution. The radius of gyration (Rg) and the length distribution function (P(r)) of OspA measured by SAXS in solution are nearly identical to the calculated ones from the crystal structure, respectively. The NMR and SAXS experiments complement each other to show that OspA including the central single‐layer β‐sheet is a stable structure in solution, and that the OspA crystal structure represents the predominant solution conformation of the protein.
Yeast methionine aminopeptidase I can utilize either Zn2+ or Co2+ as a cofactor: A case of mistaken identity?
Authors: Walker Kenneth W. , Bradshaw Ralph A.
Published: February 2009

Yeast methionine aminopeptidase I (MetAP I) is one of two enzymes in Saccharomyces cerevisiae that is responsible for cotranslational cleavage of initiator methionines. It has previously been classified as a Co2+ metalloprotease in all prokaryotic and eukaryotic forms studied. However, treatment of recombinant apo‐MetAP I with 12.5 μM Zn2+ produces an enzyme that is as active as that reconstituted with 200 μM Co2+. In the presence of physiological concentrations of reduced glutathione (GSH), Co‐MetAP I is inactive, while the activity of Zn‐MetAP I is increased more than 1.7‐fold over Zn‐MetAP I assayed in the absence of GSH. Given that the in vivo concentration of Zn2+ is at least 1,000‐fold higher than that of Co2+, and that Co2+ is insoluble in physiological concentrations of GSH, it is probable that yeast MetAP I is actually a Zn2+ metalloprotease. Furthermore, unless there are extraordinary conditions that insulate or sequester them from this reducing milieu, that have yet to be identified, there are not likely to be any cytoplasmic enzymes that use free Co2+.
Re‐examining the role of Lys67 in class C β‐lactamase catalysis
Authors: Chen Yu , McReynolds Andrea , Shoichet Brian K.
Published: February 2009

Lys67 is essential for the hydrolysis reaction mediated by class C β‐lactamases. Its exact catalytic role lies at the center of several different proposed reaction mechanisms, particularly for the deacylation step, and has been intensely debated. Whereas a conjugate base hypothesis postulates that a neutral Lys67 and Tyr150 act together to deprotonate the deacylating water, previous experiments on the K67R mutants of class C β‐lactamases suggested that the role of Lys67 in deacylation is mainly electrostatic, with only a 2‐ to 3‐fold decrease in the rate of the mutant vs the wild type enzyme. Using the Class C β‐lactamase AmpC, we have reinvestigated the activity of this K67R mutant enzyme, using biochemical and structural studies. Both the rates of acylation and deacylation were affected in the AmpC K67R mutant, with a 61‐fold decrease in kcat, the deacylation rate. We have determined the structure of the K67R mutant by X‐ray crystallography both in apo and transition state‐analog complexed forms, and observed only minimal conformational changes in the catalytic residues relative to the wild type. These results suggest that the arginine side chain is unable to play the same catalytic role as Lys67 in either the acylation or deacylation reactions catalyzed by AmpC. Therefore, the activity of this mutant can not be used to discredit the conjugate base hypothesis as previously concluded, although the reaction catalyzed by the K67R mutant itself likely proceeds by an alternative mechanism. Indeed, a manifold of mechanisms may contribute to hydrolysis in class C β‐lactamases, depending on the enzyme (wt or mutant) and the substrate, explaining why different mutants and substrates seem to support different pathways. For the WT enzyme itself, the conjugate base mechanism may be well favored.
A review about nothing: Are apolar cavities in proteins really empty?
Authors: Matthews Brian W. , Liu Lijun
Published: February 2009

Cavities within proteins that are strictly apolar typically appear to be empty. It has been suggested, however, that water molecules may be present within such cavities but are too disordered to be seen in conventional crystallographic analyses. In contrast, it is argued here that solvent mobility will be limited by the size of the cavity and for this reason high‐occupancy solvent in cavities of typical volume should be readily detectable using X‐ray crystallography. Recent experimental studies of cavity hydration are reviewed. Such studies are consistent with theoretical predictions that it is energetically unfavorable to have a single water molecule in an apolar cavity. As apolar cavities become larger, a point is reached where it is favorable to have the cavity occupied by a cluster of mutually H‐bonded water molecules. The exact size of such a cavity in a protein is yet to be verified.
Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases
Authors: Zani Marie‐Louise , Baranger Kévin , Guyot Nicolas , Dallet‐Choisy Sandrine , Moreau Thierry
Published: February 2009

The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin‐2 are endogeneous low‐molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol‐based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin‐2 variant, trappin‐2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin‐2 A62L are tight‐binding inhibitors of all three NSPs with subnanomolar Kis, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane‐bound forms of all three NSPs. The trappin‐2 A62L and elafin‐SLPI chimeras, like wild‐type elafin and trappin‐2, can be covalently cross‐linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti‐inflammatory agents.
The strong dimerization of the transmembrane domain of the fibroblast growth factor receptor (FGFR) is modulated by C‐terminal juxtamembrane residues
Authors: Peng Weng Chuan , Lin Xin , Torres Jaume
Published: February 2009

The fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR subfamily of the receptor tyrosine kinases (RTKs) involved in signaling across the plasma membrane. Generally, ligand binding leads to receptor dimerization and activation. Dimerization involves the transmembrane (TM) domain, where mutations can lead to constitutive activation in certain cancer types and also in skeletal malformations. Thus, it has been postulated that FGFR homodimerization must be inherently weak to allow regulation, a feature reminiscent of α and β integrin TM interactions. However, we show herein that in FGFR3‐TM, four C‐terminal residues, CRLR, have a profound destabilizing effect in an otherwise strongly dimerizing TM peptide. In the absence of these four residues, the dimerizing propensity of FGFR3‐TM is comparable to glycophorin, as shown using various detergents. In addition, the expected enhanced dimerization induced by the mutation associated to the Crouzon syndrome A391E, was observed only when these four C‐terminal residues were present. In the absence of these four residues, A391E was dimer‐destabilizing. Finally, using site specific infrared dichroism and convergence with evolutionary conservation data, we have determined the backbone model of the FGFR3‐TM homodimer in model lipid bilayers. This model is consistent with, and correlates with the effects of, most known pathological mutations found in FGFR‐TM.
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

mPlum 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

Nitric 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

The 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

The 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

We 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

Viruses 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

For 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

In 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

Amyloid 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

One 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

The 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

The 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

Species 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

Phosphorus 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
Management and prevention of drug‐induced major bleeding
Authors: Musumba Crispin , Pirmohamed Munir
Published: February 2009

Our series on serious ADRs focusses on rare but potentially fatal drug reactions and how to recognise and avoid them. This article considers the drugs most commonly associated with major bleeding and discusses subsequent management. Copyright © 2006 Wiley Interface Ltd
Managementof drug‐induced interstitial lung disease
Authors: Ganguli Amitava , Pirmohamed Munir
Published: February 2009

Our series on serious ADRs focusses on rare but potentially fatal drug reactions and how to recognise and avoid them. This article describes the drugs most commonly associated with interstitial lung disease and discusses subsequent management. Copyright © 2006 Wiley Interface Ltd
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

In 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.
Matrix‐assisted laser desorption/ionization mass spectrometric imaging of complete rat sections using a triple quadrupole linear ion trap
Authors: Hopfgartner Gérard , Varesio Emmanuel , Stoeckli Markus
Published: February 2009

The fast imaging of complete rat sections by matrix‐assisted laser desorption/ionization on a triple quadrupole linear ion trap is demonstrated. After administration of the pharmaceutical compound (MW = 467.4 u) at 0.5 mg/kg the parent drug could be identified in full scan mode and in the enhanced product ion spectrum mode. Furthermore, the precursor ion mode could also be used to monitor the presence of the parent drug in the tissue section. In the selected reaction monitoring mode, using a laser frequency of 1000 Hz and a rastering speed of about 18 mm/s, a targeted representative image of drug distribution in a rat section could be obtained in less than 15 min. Copyright © 2009 John Wiley & Sons, Ltd.
Modeling disease‐state transition heterogeneity through Bayesian variable selection
Authors: Healy Brian C. , Engler David
Published: February 2009

In many diseases, Markov transition models are useful in describing transitions between discrete disease states. Often the probability of transitioning from one state to another varies widely across subjects. This heterogeneity is driven, in part, by a possibly unknown number of previous disease states and by potentially complex relationships between clinical data and these states. We propose use of Bayesian variable selection in Markov transition models to allow estimation of subject‐specific transition probabilities. Our approach simultaneously estimates the order of the Markov process and the transition‐specific covariate effects. The methods are assessed using simulation studies and applied to model disease‐state transition on the expanded disability status scale (EDSS) in multiple sclerosis (MS) patients from the Partners MS Center in Boston, MA. The proposed methodology is shown to accurately identify complex covariate–transition relationships in simulations and identifies a clinically significant interaction between relapse history and EDSS history in MS patients. Copyright © 2009 John Wiley & Sons, Ltd.
Bayesian multivariate disease mapping and ecological regression with errors in covariates: Bayesian estimation of DALYs and ‘preventable’ DALYs
Authors: MacNab Ying C.
Published: February 2009

This paper presents Bayesian multivariate disease mapping and ecological regression models that take into account errors in covariates. Bayesian hierarchical formulations of multivariate disease models and covariate measurement models, with related methods of estimation and inference, are developed as an integral part of a Bayesian disability adjusted life years (DALYs) methodology for the analysis of multivariate disease or injury data and associated ecological risk factors and for small area DALYs estimation, inference, and mapping. The methodology facilitates the estimation of multivariate small area disease and injury rates and associated risk effects, evaluation of DALYs and ‘preventable’ DALYs, and identification of regions to which disease or injury prevention resources may be directed to reduce DALYs. The methodology interfaces and intersects the Bayesian disease mapping methodology and the global burden of disease framework such that the impact of disease, injury, and risk factors on population health may be evaluated to inform community health, health needs, and priority considerations for disease and injury prevention. A burden of injury study on road traffic accidents in local health areas in British Columbia, Canada, is presented as an illustrative example. Copyright © 2009 John Wiley & Sons, Ltd.
Multivariate modelling of responses to conditional items: New possibilities for latent class analysis
Authors: Spycher B. D. , Minder C. E. , Kuehni C. E.
Published: February 2009

Questionnaire data may contain missing values because certain questions do not apply to all respondents. For instance, questions addressing particular attributes of a symptom, such as frequency, triggers or seasonality, are only applicable to those who have experienced the symptom, while for those who have not, responses to these items will be missing. This missing information does not fall into the category ‘missing by design’, rather the features of interest do not exist and cannot be measured regardless of survey design. Analysis of responses to such conditional items is therefore typically restricted to the subpopulation in which they apply. This article is concerned with joint multivariate modelling of responses to both unconditional and conditional items without restricting the analysis to this subpopulation. Such an approach is of interest when the distributions of both types of responses are thought to be determined by common parameters affecting the whole population. By integrating the conditional item structure into the model, inference can be based both on unconditional data from the entire population and on conditional data from subjects for whom they exist. This approach opens new possibilities for multivariate analysis of such data. We apply this approach to latent class modelling and provide an example using data on respiratory symptoms (wheeze and cough) in children. Conditional data structures such as that considered here are common in medical research settings and, although our focus is on latent class models, the approach can be applied to other multivariate models. Copyright © 2009 John Wiley & Sons, Ltd.
How can the Yelkouan shearwater survive feral cat predation? A meta‐population structure as a solution?
Authors: Bonnaud Elsa , Bourgeois Karen , Vidal Eric , Legrand Jérôme , Le Corre Matthieu
Published: February 2009

The Yelkouan shearwater, Puffinus yelkouan, is an endangered Mediterranean endemic species of burrowing petrel threatened by feral cats. The life‐history parameters of a small population of Yelkouan shearwaters on the Mediterranean island, Port‐Cros, were studied in conjunction with the diet of feral cats, to examine the birds’ vulnerability to introduced cats. Yelkouan shearwaters were the birds most frequently found in cat scats, with 431 ± 72 birds killed per year, and predation highest during the pre‐laying period. A demographic model was created using data for P. yelkouan and for closely related shearwater species. Without cat predation, only two of four survival rate scenarios led to a mean growth rate (λ) ≥ 1. The model was constrained to have a stable population growth rate and used to predict predation scenarios compatible with the observed population stability, because the population under study has remained stable at around 180 pairs for at least 20 years despite feral cat predation. The results of assuming that the population is closed were inconsistent with the estimated mortalities due to feral cats, while it was possible to reconcile the observed numbers of breeding pairs with the observed mortalities due to cats by assuming that Port‐Cros Island is a sink sustained by immigration. This illustrates that small colonies may need to be sustained by larger ones to avoid being driven to extinction. Unfortunately, the absence of a large geographic‐scale ringing program makes the precise identification of the origin of the immigrants impossible in this case.