bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oxidized and synchrotron cleaved structures of the disulfide redox center in the N‐terminal domain of Salmonella typhimurium AhpF
Authors: Roberts Blaine R. , Wood Zachary A. , Jönsson Thomas J. , Poole Leslie B. , Karplus P. Andrew 
Published: August 2005

AbstractThe flavoprotein component (AhpF) of Salmonella typhimurium alkyl hydroperoxide reductase contains an N‐terminal domain (NTD) with two contiguous thioredoxin folds but only one redox‐active disulfide (within the sequence ‐Cys129‐His‐Asn‐Cys132‐). This active site is responsible for mediating the transfer of electrons from the thioredoxin reductase‐like segment of AhpF to AhpC, the peroxiredoxin component of the two‐protein peroxidase system. The previously reported crystal structure of AhpF possessed a reduced NTD active site, although fully oxidized protein was used for crystallization. To further investigate this active site, we crystallized an isolated recombinant NTD (rNTD); using diffraction data sets collected first at our in‐house X‐ray source and subsequently at a synchrotron, we showed that the active site disulfide bond (Cys129–Cys132) is oxidized in the native crystals but becomes reduced during synchrotron data collection. The NTD disulfide bond is apparently particularly sensitive to radiation cleavage compared with other protein disulfides. The two data sets provide the first view of an oxidized (disulfide) form of NTD and show that the changes in conformation upon reduction of the disulfide are localized and small. Furthermore, we report the apparent pKa of the active site thiol to be ∼5.1, a relatively low pKa given its redox potential (∼265 mV) compared with most members of the thioredoxin family.
An improved experimental system for determining small folding entropy changes resulting from proline to alanine substitutions
Authors: Street Timothy O. , Bradley Christina Marchetti , Barrick Doug 
Published: August 2005

AbstractChanges in protein stability can be achieved by making substitutions that increase or decrease the available conformations of the unfolded protein without altering the conformational freedom of the folded protein. Matthews and coworkers (1987) proposed that proline to alanine (P → A) substitution would achieve this type of entropic destabilization. By comparing the Ramachandran area associated with alanine and proline residues, Matthews et al. estimated the unfolding entropy change resulting from P → A substitution to be 4.8 cal mol−1K−1. Although such an entropy difference would produce a substantial free energy change, accurately resolving such free energy changes into entropic and enthalpic components has been difficult. Here, we attempt to quantify the unfolding entropy change produced by P → A substitution by amplifying the effect through multiple substitutions, and by decreasing the uncertainty in determining the unfolding entropy. Variants of a repeat protein, theDrosophilaNotch ankyrin domain, were constructed with a varying number of P → A substitutions at structurally conserved positions. Unfolding entropy values of the variants were determined from free energy measurements taken over a common temperature range using chemical denaturation. Our findings confirm the prediction that increasing the number of proline residues present in similar local environments increases the unfolding entropy. The average value of this increase in unfolding entropy is 7.7 ± 4.2 cal mol−1K−1, which is within error of the value estimated by Matthews et al. (1987).
Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering
Authors: Deuschle Karen , Okumoto Sakiko , Fehr Marcus , Looger Loren L. , Kozhukh Leonid , Frommer Wolf B. 
Published: August 2005

AbstractA family of genetically‐encoded metabolite sensors has been constructed using bacterial periplasmic binding proteins (PBPs) linearly fused to protein fluorophores. The ligand‐induced conformational change in a PBP allosterically regulates the relative distance and orientation of a fluorescence resonance energy transfer (FRET)‐compatible protein pair. Ligand binding is transduced into a macroscopic FRET observable, providing a reagent for in vitro and in vivo ligand‐measurement and visualization. Sensors with a higher FRET signal change are required to expand the dynamic range and allow visualization of subtle analyte changes under high noise conditions. Various observations suggest that factors other than inter‐fluorophore separation contribute to FRET transfer efficiency and the resulting ligand‐dependent spectral changes. Empirical and rational protein engineering leads to enhanced allosteric linkage between ligand binding and chromophore rearrangement; modifications predicted to decrease chromophore rotational averaging enhance the signal change, emphasizing the importance of the rotational freedom parameter κ2 to FRET efficiency. Tighter allosteric linkage of the PBP and the fluorophores by linker truncation or by insertion of chromophores into the binding protein at rationally designed sites gave rise to sensors with improved signal change. High‐response sensors were obtained with fluorescent proteins attached to the same binding PBP lobe, suggesting that indirect allosteric regulation during the hinge‐bending motion is sufficient to give rise to a FRET response. The optimization of sensors for glucose and glutamate, ligands of great clinical interest, provides a general framework for the manipulation of ligand‐dependent allosteric signal transduction mechanisms.
Studies of helix fraying and solvation using 13C′ isotopomers
Authors: Fesinmeyer R. Matthew , Peterson Eric S. , Dyer R. Brian , Andersen Niels H. 
Published: August 2005

AbstractBoth NMR and IR studies of carbonyl (13C′) isotopomers of designed helices can provide residue‐level details regarding the fractional occurrence and melting behavior of helical ϕ/ψ angles along the sequence of helical peptides, details that cannot be obtained from CD or 1H‐NMR studies. We have studied a classic series of helical models, Ac‐YGG‐(KAXAA)3K‐NH2 (X=A,V), in both aqueous and helix‐favoring media containing fluoroalcohol cosolvents, including a solvent system allowing the observation of cold denaturation. These studies confirmed the strong N‐capping associated with this sequence and revealed more extensive C‐terminal fraying than that calculated using current helicity prediction algorithms. In the X=A series, the central residues are somewhat resistant to thermal melting; it instead occurs predominantly at the frayable C terminus. For the X=V series under cold‐denaturing conditions, the temperature of maximal helicity is not uniform along the sequence and both solvated and nonsolvated helical alanine sites (13C=O stretches at 1592 cm−1 and 1615 cm−1, respectively) are apparent. Correlation between the two spectroscopies employed yielded the intriguing observation that the valine side chain is able to desolvate the i − 4 amide in short monomeric helices. In addition, we report further measurements of the temperature dependence of alanine statistical coil chemical shifts, the temperature dependence of the 13C chemical shift of urea (employed as chemical shift reference), and a useful formula for converting 13C′ shifts into fractional helicities.
Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis
Authors: Pál Gábor , Fong Shun‐Yin , Kossiakoff Anthony A. , Sidhu Sachdev S. 
Published: August 2005

AbstractCombinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost‐effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine‐scanning approach was expanded with two additional strategies, serine‐ and homolog‐scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor‐binding sites (site 1) of wild‐type hGH (hGHwt) and of an affinity‐improved variant (hGHv) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine‐ and homolog‐scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild‐type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild‐type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine‐scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGHv. The serine and homolog‐scanning results expand upon and complement the alanine‐scanning results and provide additional data on the robustness of the high affinity receptor‐binding site of hGH.
Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines
Authors: García Javier , Puentes Alvaro , Rodríguez Luis , Ocampo Marisol , Curtidor Hernando , Vera Ricardo , Lopez Ramses , Valbuena John , Cortes Jimena , Vanegas Magnolia , Barrero Carlos , Patarroyo Manuel A. , Urquiza Mauricio , Patarroyo Manuel E. 
Published: August 2005

AbstractThe gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT‐PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25‐kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20‐amino‐acid‐long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross‐linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.
Rapid and accurate structure determination of coiled‐coil domains using NMR dipolar couplings: Application to cGMP‐dependent protein kinase Iα
Authors: Schnell Jason R. , Zhou Guo‐Ping , Zweckstetter Markus , Rigby Alan C. , Chou James J. 
Published: August 2005

AbstractCoiled‐coil motifs play essential roles in protein assembly and molecular recognition, and are therefore the targets of many ongoing structural and functional studies. However, owing to the dynamic nature of many of the smaller coiled‐coil domains, crystallization for X‐ray studies is very challenging. Determination of elongated structures using standard NMR approaches is inefficient and usually yields low‐resolution structures due to accumulation of small errors over long distances. Here we describe a solution NMR approach based on residual dipolar couplings (RDCs) for rapid and accurate structure determination of coiled‐coil dimers. Using this approach, we were able to determine the high‐resolution structure of the coiled‐coil domain of cGMP‐dependent protein kinase Iα, a protein of previously unknown structure that is critical for physiological relaxation of vascular smooth muscle. This approach can be extended to solve coiled‐coil structures with higher order assemblies.
A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: Structural determinants of redox potential
Authors: Alagaratnam Sharmini , van Pouderoyen Gertie , Pijning Tjaard , Dijkstra Bauke W. , Cavazzini Davide , Rossi Gian Luigi , Van Dongen Walter M.A.M. , van Mierlo Carlo P.M. , van Berkel Willem J. H. , Canters Gerard W. 
Published: August 2005

AbstractFlavodoxin II from Azotobacter vinelandii is a “long‐chain” flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three‐dimensional structure determined to a resolution of 2.25 Å by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five‐stranded parallel β‐sheet flanked on either side by α‐helices. An eight‐residue insertion, compared with other long‐chain flavodoxins, forms a short 310 helix preceding the start of the α3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent‐accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.
A bioinformatic tool for analysis of EST transcript abundance during infection‐related development by Magnaporthe grisea
Authors: SOANES DARREN M. , TALBOT NICHOLAS J. 
Published: August 2005

SUMMARYInformation regarding the levels of mRNA transcript abundance under different conditions, or in specific tissue types, can be obtained by analysis of the frequency of EST sequences in randomly sequenced cDNA libraries. Here we report a bioinformatics tool, which provides a means of identifying genes that are differentially expressed during pathogenesis‐related development by the rice blast fungus Magnaporthe grisea. A total of 31 534 M. grisea ESTs were obtained from dbEST at NCBI, clustered into 8821 unique sequences (unisequences) and manually annotated. Transcript profiles were then calculated for 958 unigenes identified from eight different cDNA libraries. The data were integrated into the Consortium for Functional Genomics of Microbial Eukaryotes (COGEME) database (http://cogeme.ex.ac.uk/) and a web‐based front end was designed to allow users to access and interrogate the generated datasets.
A gene with domains related to transcription regulation is required for pathogenicity in Colletotrichum acutatum causing Key lime anthracnose
Authors: CHEN HUI‐QIN , CAO LIHUA , DEKKERS KATHERINE L. , ROLLINS JEFFREY A. , KO NAN J. , TIMMER L. W. , CHUNG KUANG‐REN 
Published: August 2005

SUMMARY Colletotrichum acutatum causes Key lime anthracnose (KLA) and postbloom fruit drop (PFD) of citrus. We utilized restriction enzyme‐mediated integration (REMI) mutagenesis to produce six non‐pathogenic mutants from a KLA isolate after screening 1064 transformants on detached Key lime leaves. Subsequently, a gene designated KLAP1 (Key Lime Anthracnose Pathogenicity) was identified from one of the mutants and was demonstrated genetically to be required for pathogenicity to Key lime leaves. The predicted polypeptide encoded by KLAP1 contains a cAMP and cGMP‐dependent protein kinase phosphorylation site, and two RGD (Arg–Gly–Asp) cell attachment sequences, a bipartite nuclear targeting sequence, a fungal G‐protein alpha subunit signature, a putative metal‐binding zinc finger (Cys2His2) and a putative HMG‐I/Y (‘high mobility group’ non‐histone chromatin protein encoding genes) DNA‐binding domain (A+T hook), suggesting that KLAP1 may function as a transcription activator in C. acutatum. Sequences homologous to KLAP1 were detected in most C. acutatum isolates examined, and similarity was found in several classes of fungi, animals, plants and bacteria, indicating that KLAP1 is a putative, uncharacterized, conserved transcription activator in fungi. Targeted gene disruption of KLAP1 yielded mutants that were blocked in the penetration stage and were completely defective in pathogenicity on Key lime leaves, but remained pathogenic to flower petals. Complementation of a klap1‐null mutant with a full‐length KLAP1 gene clone restored complete ability to incite lesions on Key lime. The results indicate that KLAP1 is an important pathogenicity factor in C. acutatum.
Microbial avirulence determinants: guided missiles or antigenic flak?
Authors: SKAMNIOTI PARASKEVI , RIDOUT CHRISTOPHER J. 
Published: August 2005

SUMMARYAvirulence (avr) determinants are incompatibility factors which elicit host plant defence responses in a gene‐for‐gene manner. They are produced by fungi, bacteria and viruses, and their recognition by resistance genes has been extensively studied for decades. But why should a microbe keep a molecule that allows it to be recognized? One argument is that avr genes perform some essential function and must be kept despite giving the pathogen away. Many bacterial avr determinants have been shown to be effectors, which contribute to virulence and aggressiveness. If this were always the case, mutants lacking these essential molecules would be at a serious disadvantage. Some disadvantage has been shown for a small number, but for the majority there is no effect on virulence. This has been explained by functional redundancy for bacterial and fungal avr determinants, with other molecules compensating for the deletion of these essential genes. However, this argument is counter‐intuitive because by definition these individual genes are no longer essential; so why keep them? With increasing numbers of avr genes being identified, efforts to elucidate their function are increasing. In this review, we take stock of the accumulating literature, and consider what the real function of avr determinants might be.
Metabolic and stress adaptation by Mycosphaerella graminicola during sporulation in its host revealed through microarray transcription profiling
Authors: KEON JOHN , RUDD JASON JAMES , ANTONIW JOHN , SKINNER WENDY , HARGREAVES JOHN , HAMMOND‐KOSACK KIM 
Published: August 2005

SUMMARYPathogenic microbes must successfully adapt to the host environment, acquiring nutrients and tolerating immune/defence responses. Studies on host–pathogen interactions at the transcriptome level have predominantly investigated host responses. Here we present a broad‐scale transcriptional analysis on a fungal pathogen during sporulation within its host environment. Septoria leaf blotch is an important fungal disease of cultivated wheat and is caused by the ascomycete fungus Septoria tritici (teleomorph Mycosphaerella graminicola). A cDNA microarray containing 2563 unigenes was generated and then used to compare fungal nutrition and development in vitro under nutrient‐rich and nutrient‐limiting conditions and in vivo at a late stage of plant infection. The data obtained provided clear insights into metabolic adaptation in all three conditions and an elevated stress adaptation/tolerance specifically in the host environment. We conclude that asexual sporulation of M. graminicola during the late stage of plant infection occurs in a rich nutritional environment involving adaptation to stresses imposed in part by the presence of reactive oxygen species.
Multiple defence signals induced by Erwinia carotovora ssp. carotovora elicitors in potato
Authors: MONTESANO MARCOS , BRADER GÜNTER , PONCE DE LEÓN INÉS , PALVA E. TAPIO 
Published: August 2005

SUMMARYSignal pathways involved in Solanum tuberosum–Erwinia carotovora ssp. carotovoraSCC3193 interaction were characterized. To this end, the concentration of several signal molecules implicated in plant defence such as ethylene (ET), jasmonates (JA) and salicylic acid (SA) were measured in potato plants treated by cell‐free culture filtrates (CF) from E. c. carotovoraSCC3193. Furthermore, the presence of other potential signalling compounds such as cinnamic acid (CA) and related aromatic compounds was screened in the elicitor‐treated plants. The activity of these signal compounds as inducers of defence‐related genes such as drd‐1 (a defence‐related alcohol dehydrogenase), pinII (proteinase inhibitor II), chtB4 (basic chitinase) and chtA2 (acidic chitinase) was characterized. The results demonstrate that ET, JA and CA accumulate in potato tissues in response to CF. These signal molecules were shown to induce differential expression of drd‐1, pinII, chtB4 and chtA2. Our data suggest that in addition to ET and JA, CA and possibly other aromatic compounds also may play a role in defence signalling in potato.
Organic matter characteristics and distribution in Ferralsol profiles of a climosequence in southern Brazil
Authors: Dalmolin R. S. D. , Gonçalves C. N. , Dick D. P. , Knicker H. , Klamt E. , Kögel‐Knabner I. 
Published: August 2005

SummaryWe studied the impact of climate, soil texture and iron mineralogy on the content and composition of organic matter in Ferralsols along a climosequence in southern Brazil. We characterized the organic matter by solid‐state cross‐polarization magic angle spinning (CPMAS) 13C nuclear magnetic resonance (NMR) spectroscopy. The organic matter content in the Ferralsols increased from the lowest to the highest sites (440–950 m altitude) as a result of the increase in humidity and the decrease in temperature. This influence was more pronounced in the heavy clayey Ferralsols, suggesting that the accumulation of organic matter was enhanced by organo‐mineral interactions. Iron oxides contributed to the stabilization of the organic matter in these soils, which was dominated by O–alkyl C structures followed by aromatic and alkyl C groups. The aromatic C contribution to the total organic C increases with depth at the expense of O–alkyl C, regardless of the site altitude and climate. Although the impact of the environment on the amount of organic matter and its chemical composition is confirmed, our results clearly show that this is so only for the surface horizons. With increasing soil depth the climatic influence decreased, showing that other factors become more important for the accumulation of organic matter.