Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein.
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Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation. (+info)
Crystal structure of a DNA.RNA hybrid duplex with a polypurine RNA r(gaagaagag) and a complementary polypyrimidine DNA d(CTCTTCTTC).
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DNA.RNA hybrid duplexes are substrates of RNase H and reverse transcriptase. The crystal structure of a hybrid duplex, d(5'-CTCTTCTTC-3').r(5'-gaagaagag-3') (the uppercase letters indicate DNA and lowercase letters RNA), with a polypurine RNA strand and a complementary DNA strand has been determined at 1.8 A resolution. The structure was refined first at 1.9 A by XPLOR and subsequently by CNS at 1.8 A. The hybrid is found in a standard A-form conformation with all the sugars in the C3'-endo puckering. The 5'-terminal base dC of the DNA strand was clearly visible in the electron density map of the present structure, in contrast to the previously reported structure d(TTCTTBr(5)CTTC).r(gaagaagaa) where the 5'-terminal base dT was not visible, leaving the terminal rA unpaired. Thus, the comparison of the terminal base pairs, C.g versus T.a, in the two hybrid crystal structures provides information on the stability of these base pairs in hydrogen bonding (three versus two) and base stacking interactions. The differences in the terminal base pairs produce different kinks in the two structures. Minor groove widening is observed in the present structure at a distinctive kink in the lower half of the duplex, in contrast to the small widening of the minor groove and a very slight bend in the upper half of the T.a structure. (+info)
Analysis of structural and physico-chemical parameters involved in the specificity of binding between alpha-amylases and their inhibitors.
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Enzyme-inhibitor specificity was studied for alpha-amylases and their inhibitors. We purified and cloned the cDNAs of two different alpha-amylase inhibitors from the common bean (Phaseolus vulgaris) and have recently cloned the cDNA of an alpha-amylase of the Mexican bean weevil (Zabrotes subfasciatus), which is inhibited by alpha-amylase inhibitor 2 but not by alpha-amylase inhibitor 1. The crystal structure of AI-1 complexed with pancreatic porcine alpha-amylase allowed us to model the structure of AI-2. The structure of Zabrotes subfasciatus alpha-amylase was modeled based on the crystal structure of Tenebrio molitor alpha-amylase. Pairwise AI-1 and AI-2 with PPA and ZSA complexes were modeled. For these complexes we first identified the interface forming residues. In addition, we identified the hydrogen bonds, ionic interactions and loss of hydrophobic surface area resulting from complex formation. The parameters we studied provide insight into the general scheme of binding, but fall short of explaining the specificity of the inhibition. We also introduce three new tools-software packages STING, HORNET and STINGPaint-which efficiently determine the interface forming residues and the ionic interaction data, the hydrogen bond net as well as aid in interpretation of multiple sequence alignment, respectively. (+info)
Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus.
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The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 and His285 as the putative members of the catalytic triad. In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose structure has been recently elucidated. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances. The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the [(3)H]diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG. (+info)
Specific RNA protease inhibitors from in vitro selection.
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RNA aptamers that bind to and inhibit the proteolytic activity of subtilisin BPN' are selected in vitro from pools of random RNA. The RNAs in vitro transcribed from the isolated clones show highly specific inhibition toward the microbial serine proteases. From the sequences of the isolated clones, a C/A-rich sequence was obtained. The kinetic features of the common C/A-rich sequence will be discussed. (+info)
Three-dimensional facial growth studied by optical surface scanning.
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The objective of the investigation was to study the three-dimensional growth of the face, and to examine the hypothesis that there are three-dimensional differences between the faces of boys and girls. The subjects comprised 132 British Caucasians aged 5-10 years measured by optical surface scanning in this cross-sectional study. Average scans for each age and sex subgroup were superimposed to assess the differences with age and sex. Males were generally larger than females. The greatest difference was between the facial heights and the least in the mid-facial dimensions. The face height of both sexes increased by an average of 3-4 mm annually. Mid-face prominence and width altered little. Mandibular width increased by 1-3 mm a year, rising to 3-5 mm in some years at the inferior areas of the mandibular region. Mandibular prominence also increased. Nose height and prominence and alar base width increased by 2 mm per year on average. Dorsum width changed little. Boys were generally larger than girls. Growth in facial height was greatest. Mid-face prominence and width changed little with age, whilst the prominence and width of the lower face increased more. Nasal prominence and alar base width increased at most ages. Dimensions changed more than reported by cephalometric studies, possibly as this study included the soft tissues. Refereed Scientific Paper (+info)
LabPatch, an acquisition and analysis program for patch-clamp electrophysiology.
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An acquisition and analysis program, "LabPatch," has been developed for use in patch-clamp research. LabPatch controls any patch-clamp amplifier, acquires and records data, runs voltage protocols, plots and analyzes data, and connects to spreadsheet and database programs. Controls within LabPatch are grouped by function on one screen, much like an oscilloscope front panel. The software is mouse driven, so that the user need only point and click. Finally, the ability to copy data to other programs running in Windows 95/98, and the ability to keep track of experiments using a database, make LabPatch extremely versatile. The system requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analog converter, and a patch-clamp amplifier. LabPatch is available free of charge at http://www.fhs.mcmaster.ca/huizinga/. (+info)
Graphical tools for comparative genome analysis.
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Visualization of data is important for many data-rich disciplines. In biology, where data sets are becoming larger and more complex, graphical analysis is felt to be ever more pertinent. Although some patterns and trends in data sets may only be determined by sophisticated computational analysis, viewing data by eye can provide us with an extraordinary amount of information in an instant. Recent advances in bioinformatic technologies allow us to link graphical tools to data sources with ease, so we can visualize our data sets dynamically. Here, an overview of graphical software tools for comparative genome analysis is given, showing that a range of simple tools can provide us with a powerful view of the differences and similarities between genomes. (+info)