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Monomeric Protein (monomeric + protein)
Selected AbstractsCharacterization of Mycobacterium tuberculosis nicotinamidase/pyrazinamidaseFEBS JOURNAL, Issue 4 2008Hua Zhang The nicotinamidase/pyrazinamidase (PncA) of Mycobacterium tuberculosis is involved in the activation of the important front-line antituberculosis drug pyrazinamide by converting it into the active form, pyrazinoic acid. Mutations in the pncA gene cause pyrazinamide resistance in M. tuberculosis. The properties of M. tuberculosis PncA were characterized in this study. The enzyme was found to be a 20.89 kDa monomeric protein. The optimal pH and temperature of enzymatic activity were pH 7.0 and 40 °C, respectively. Inductively coupled plasma-optical emission spectrometry revealed that the enzyme was an Mn2+/Fe2+ -containing protein with a molar ratio of [Mn2+] to [Fe2+] of 1 : 1; furthermore, the external addition of either type of metal ion had no apparent effect on the wild-type enzymatic activity. The activity of the purified enzyme was determined by HPLC, and it was shown that it possessed similar pyrazinamidase and nicotinamidase activity, by contrast with previous reports. Nine PncA mutants were generated by site-directed mutagenesis. Determination of the enzymatic activity and metal ion content suggested that Asp8, Lys96 and Cys138 were key residues for catalysis, and Asp49, His51, His57 and His71 were essential for metal ion binding. Our data show that M. tuberculosis PncA may bind metal ions in a manner different from that observed in the case of Pyrococcus horikoshii PncA. [source] Identification of RNase HII from psychrotrophic bacterium, Shewanella sp.FEBS JOURNAL, Issue 10 2006SIB1 as a high-activity type RNase H The gene encoding RNase HII from the psychrotrophic bacterium, Shewanella sp. SIB1 was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RNase HII is a monomeric protein with 212 amino acid residues and shows an amino acid sequence identity of 64% to E. coli RNase HII. The enzymatic properties of SIB1 RNase HII, such as metal ion preference, pH optimum, and cleavage mode of substrate, were similar to those of E. coli RNase HII. SIB1 RNase HII was less stable than E. coli RNase HII, but the difference was marginal. The half-lives of SIB1 and E. coli RNases HII at 30 °C were ,,30 and 45 min, respectively. The midpoint of the urea denaturation curve and optimum temperature of SIB1 RNase HII were lower than those of E. coli RNase HII by ,,0.2 m and ,,5 °C, respectively. However, SIB1 RNase HII was much more active than E. coli RNase HII at all temperatures studied. The specific activity of SIB1 RNase HII at 30 °C was 20 times that of E. coli RNase HII. Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII represent low- and high-activity type RNases H, respectively, in SIB1. In contrast, RNases HI and HII represent high- and low-activity type RNases H, respectively, in E. coli. We propose that bacterial cells usually contain low- and high-activity type RNases H, but these types are not correlated with RNase H families. [source] Bacterial IscU is a well folded and functional single domain proteinFEBS JOURNAL, Issue 11 2004Salvatore Adinolfi Iron,sulfur clusters are widely represented in most organisms, but the mechanism of their formation is not fully understood. Of the two main proteins involved in cluster formation, NifS/IscS and NifU/IscU, only the former has been well studied from a structural point of view. Here we report an extensive structural characterization of Escherichia coli IscU. We show by a variety of physico-chemical techniques that E. coli IscU construct can be expressed to high purity as a monomeric protein, characterized by an ,, fold with high ,-helix content. The high melting temperature and the reversibility of the thermal unfolding curve (as measured by CD spectroscopy) hint at a well ordered stable fold. The excellent dispersion of cross peaks in the 1H- 15N correlation spectrum is consistent with these observations. Monomeric E. coli IscU is able to provide a scaffold for Iron,sulfur cluster assembly, but has no direct interaction with either Fe(II) or Fe(III) ions, suggesting the need of further partners to achieve a stable interaction. [source] A structural proteomics filter: prediction of the quaternary structural type of hetero-oligomeric proteins on the basis of their sequencesJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 6 2007Oliviero Carugo A protein chain can correspond to a monomeric protein or it can form, together with other chains, oligomeric assemblies, which can be either homo-oligomers or hetero-oligomers. In the latter case, the three-dimensional structure of the single protein chain is unlikely to be determined, since it will probably be difficult to express and crystallize. A computational method is presented here that allows one to predict if a chain participates in hetero-oligomeric assemblies, on the basis of its amino acid composition, with accuracy close to 80%. Such a technique should improve the success rate of structural biology projects. [source] Mimicry of dimerization by synthetic peptides designed to target homologous regions of proteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2003Donard S. Dwyer Abstract Rapid progress in sequencing various genomes has highlighted the need for the development of biochemical reagents for the detection of thousands of expressed gene products. The magnitude of this detection problem exceeds current technical capabilities. In an attempt to address this shortcoming, a novel approach has been developed called mimicry of dimerization. Peptide tags have been designed to bind to a specific region of parvalbumin on the basis of amino acid sequence homology with this segment. Multivalent ligands were produced by coupling the synthetic peptides to activated dextran polymers and binding was assessed by chemiluminescence of enhanced avidity reactions using a high density of target protein at the binding surface. Binding of the peptide ligands to parvalbumin was strongest under assay conditions that enriched for native monomeric protein and was affected by pH, temperature and solvent conditions. The results suggest that it should be possible to develop specific reagents for tagging proteins on the basis of sequence and secondary structure information. [source] Peroxiredoxin Q of Arabidopsis thaliana is attached to the thylakoids and functions in context of photosynthesis,THE PLANT JOURNAL, Issue 6 2006Petra Lamkemeyer Summary Peroxiredoxin Q (Prx Q) is one out of 10 peroxiredoxins encoded in the genome of Arabidopsis thaliana, and one out of four that are targeted to plastids. Peroxiredoxin Q functions as a monomeric protein and represents about 0.3% of chloroplast proteins. It attaches to the thylakoid membrane and is detected in preparations enriched in photosystem II complexes. Peroxiredoxin Q decomposes peroxides using thioredoxin as an electron donor with a substrate preference of H2O2 > cumene hydroperoxide , butyl hydroperoxide , linoleoyl hydroperoxide and insignificant affinity towards complex phospholipid hydroperoxide. Plants with decreased levels of Prx Q did not have an apparently different phenotype from wildtype at the plant level. However, similar to antisense 2-cysteine (2-Cys) Prx plants [Baier, M. et al. (2000)Plant Physiol., 124, 823,832], Prx Q-deficient plants had a decreased sensitivity to oxidants in a leaf slice test as indicated by chlorophyll a fluorescence measurements. Increased fluorescence ratios of photosystem II to I at 77 K and modified transcript levels of plastid- and nuclear-encoded proteins show that regulatory mechanisms are at work to compensate for the lack of Prx Q. Apparently Prx Q attaches to photosystem II and has a specific function distinct from 2-Cys peroxiredoxin in protecting photosynthesis. Its absence causes metabolic changes that are sensed and trigger appropriate compensatory responses. [source] Design and Characterisation of an Artificial DNA-Binding CytochromeCHEMBIOCHEM, Issue 7 2004D. Dafydd Jones Dr. Abstract We aim to design novel proteins that link specific biochemical binding events, such as DNA recognition, with electron transfer functionality. We want these proteins to form the basis of new molecules that can be used for templated assembly of conducting cofactors or for thermodynamically linking DNA binding with cofactor chemistry for nanodevice applications. The first examples of our new proteins recruit the DNA-binding basic helix region of the leucine zipper protein GCN4. This basic helix region was attached to the N and C termini of cytochrome b562(cyt b562) to produce new, monomeric, multifunctional polypeptides. We have fully characterised the DNA and haem-binding properties of these proteins, which is a prerequisite for future application of the new molecules. Attachment of a single basic helix of GCN4 to either the N or C terminus of the cytochrome does not result in specific DNA binding but the presence of DNA-binding domains at both termini converts the cytochrome into a specific DNA-binding protein. Upon binding haem, this chimeric protein attains the spectral characteristics of wild-type cyt b562. The three forms of the protein, apo, oxidised holo and reduced holo, all bind the designed (ATGAcgATGA) target DNA sequence with a dissociation constant, KD, of approximately 90 nM. The protein has a lower affinity (KDca. 370 nM) for the wild-type GCN4 recognition sequence (ATGAcTCAT). The presence of only half the consensus DNA sequence (ATGAcgGGCC) shifts the KDvalue to more than 2500 nM and the chimera does not bind specifically to DNA sequences with no target recognition sites. Ultracentrifugation revealed that the holoprotein,DNA complex is formed with a 1:1 stoichiometry, which indicates that a higher-order protein aggregate is not responsible for DNA binding. Mutagenesis of a loop linking helices 2 and 3 of the cytochrome results in a chimera with a haem-dependent DNA binding affinity. This is the first demonstration that binding of a haem group to a designed monomeric protein can allosterically modulate the DNA binding affinity. [source] On the Temperature,Pressure Free-Energy Landscape of ProteinsCHEMPHYSCHEM, Issue 4 2003Revanur Ravindra Dr. Abstract We studied the thermodynamic stability of a small monomeric protein, staphylococcal nuclease (Snase), as a function of both temperature and pressure, and expressed it as a 3D free-energy surface on the p,T -plane using a second-order Taylor expansion of the Gibbs free-energy change ,G upon unfolding. We took advantage of a series of different techniques (small-angle Xray scattering, Fourier-transform infrared spectroscopy, differential thermal analysis, pressure perturbation calorimetry and densitometry) in the evaluation of the conformation of the protein and in evaluating the changes in the thermodynamic parameters upon unfolding, such as the heat capacity, enthalpy, entropy, volume, isothermal compressibility and expansivity. The calculated results of the free-energy landscape of the protein are in good agreement with experimental data of the p,T -stability diagram of the protein over a temperature range from 200 to 400 K and at pressures from ambient pressure to 4000 bar. The results demonstrate that combined temperature,pressure-dependent studies can help delineate the free-energy landscape of proteins and hence help elucidate which features and thermodynamic parameters are essential in determining the stability of the native conformational state of proteins. The approach presented may also be used for studying other systems with so-called re-entrant or Tamman loop-shaped phase diagrams. [source] High epitope density in a single protein molecule significantly enhances antigenicity as well as immunogenicity: a novel strategy for modern vaccine development and a preliminary investigation about B,cell discrimination of monomeric proteinsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2005Wanli Liu Abstract Although early studies have shown a close correlation between epitope density and epitope-specific humoral immune responses, few attempts have been made to quantitatively compare the antigenic and immunogenic differences between protein molecules bearing low or high degrees of epitope density, nor have studies quantitatively investigated the mechanism of B,cell discrimination of monomeric antigens. In this study, we prepared glutathione S-transferase (GST) fusion proteins bearing various copies of the M2e epitope from the influenza virus M2,protein [GST-(M2e)8, GST-(M2e)4 and GST-(M2e)1], which were used to detect and compare the real-time kinetic binding with M2e-specific mAb by surface plasma resonance. Our data show clearly that fusion proteins bearing higher M2e epitope density resulted in higher average avidity for M2e-specific mAb. Furthermore, it was observed that fusion proteins bearing high M2e epitope density could induce polyclonal antibodies (pAb) with enhanced an average affinity constant (KA) for M2e epitope peptide compared to fusion proteins bearing low epitope density. The average KA of pAb induced by GST-(M2e)8 (3.08 × 108,M,1 or 9.96 × 108,M,1) was up to two orders of magnitude greater than the average KA of pAb induced by GST-(M2e)1 (2.00 × 106,M,1 or 3.43 × 106,M,1). Thus, the data presented here demonstrate that high epitope density in a single protein molecule significantly enhances antigenicity and immunogenicity. These findings enrich our knowledge of how epitope density might relate to the recognition, activation and antibody production processes of epitope-specific immature B,cells. [source] Differences in polymeric proteins among grains in spring wheat spikesJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 4 2006A Andersson Abstract A uniform amount and size distribution of polymeric proteins within grains in a spike might determine the stability of wheat quality. Two cultivars were grown to maturity in solution culture in a climate chamber. Nitrogen (N) in the form of nitrate was added daily and replaced with 15N before harvest. Plants were harvested during grain development. Protein composition and relationships of labelled N in grains from different spikelets within the spike were determined. Higher percentages of large unextractable polymeric proteins (%-LUPP) and total unextractable polymeric proteins (%-TUPP) were found in the lower- and uppermost spikelets in the spike compared with the middle ones for cv. WL, but not for cv. Sport. Both cultivars showed variations in the percentage of large unextractable monomeric proteins (%-LUMP) and total SDS-extractable protein (Tote) in the spikelets within the spike. The amount of total SDS-unextractable protein (Totu) did not vary for either of the cultivars. The spikelets within the spike showing high and low %-LUMP and Tote at maturity showed a similar behaviour shortly after flowering in cv. WL, but not in cv. Sport. The N concentration of SDS and sonicated extracts varied along the spikelets of the spike for both cultivars. The atom-% excess 15N decreased in cv. Sport SDS-extractable and -unextractable proteins and cv. WL albumins + globulins, gliadins and glutenins from grains at different spikelet positions along the spike. Copyright © 2005 Society of Chemical Industry [source] The role of entropy and polarity in intermolecular contacts in protein crystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Marcin Cie The integrity and X-ray diffraction quality of protein crystals depend on the three-dimensional order of relatively weak but reproducible intermolecular contacts. Despite their importance, relatively little attention has been paid to the chemical and physical nature of these contacts, which are often regarded as stochastic and thus not different from randomly selected protein surface patches. Here, logistic regression was used to analyze crystal contacts in a database of 821 unambiguously monomeric proteins with structures determined to 2.5,Å resolution or better. It is shown that the propensity of a surface residue for incorporation into a crystal contact is not a linear function of its solvent-accessible surface area and that amino acids with low exposed surfaces, which are typically small and hydrophobic, have been underestimated with respect to their contact-forming potential by earlier area-based calculations. For any given solvent-exposed surface, small and hydrophobic residues are more likely to be involved in crystal contacts than large and charged amino acids. Side-chain entropy is the single physicochemical property that is most negatively correlated with the involvement of amino acids in crystal contacts. It is also shown that crystal contacts with larger buried surfaces containing eight or more amino acids have cores that are depleted of polar amino acids. [source] A Designed Well-Folded Monomeric Four-Helix Bundle Protein Prepared by Fmoc Solid-Phase Peptide Synthesis and Native Chemical Ligation,CHEMISTRY - A EUROPEAN JOURNAL, Issue 5 2006Gunnar T. Dolphin Dr. Abstract The design and total chemical synthesis of a monomeric native-like four-helix bundle protein is presented. The designed protein, GTD-Lig, consists of 90 amino acids and is based on the dimeric structure of the de novo designed helix-loop-helix GTD-43. GTD-Lig was prepared by the native chemical ligation strategy and the fragments (45 residues long) were synthesized by applying standard fluorenylmethoxycarbonyl (Fmoc) chemistry. The required peptide,thioester fragment was prepared by anchoring the free ,-carboxy group of Fmoc-Glu-allyl to the solid phase. After chain elongation the allyl moiety was orthogonally removed and the resulting carboxy group was functionalized with a glycine,thioester followed by standard trifluoroacetic acid (TFA) cleavage to produce the unprotected peptide,thioester. The structure of the synthetic protein was examined by far- and near-UV circular dichroism (CD), sedimentation equilibrium ultracentrifugation, and NMR and fluorescence spectroscopy. The spectroscopic methods show a highly helical and native-like monomeric protein consistent with the design. Heat-induced unfolding was studied by tryptophan absorbance and far-UV CD. The thermal unfolding of GTD-Lig occurs in two steps; a cooperative transition from the native state to an intermediate state and thereafter by noncooperative melting to the unfolded state. The intermediate exhibits the properties of a molten globule such as a retained native secondary structure and a compact hydrophobic core. The thermodynamics of GuHCl-induced unfolding were evaluated by far-UV CD monitoring and the unfolding exhibited a cooperative transition that is well-fitted by a two-state mechanism from the native to the unfolded state. GTD-Lig clearly shows the characteristics of a native protein with a well-defined structure and typical unfolding transitions. The design and synthesis presented herein is of general applicability for the construction of large monomeric proteins. [source] | ||||||||||