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Isolated Protein (isolated + protein)
Selected AbstractsBinding of ligands originates small perturbations on the microscopic thermodynamic properties of a multicentre redox proteinFEBS JOURNAL, Issue 9 2005Carlos A. Salgueiro NMR and visible spectroscopy coupled to redox measurements were used to determine the equilibrium thermodynamic properties of the four haems in cytochrome c3 under conditions in which the protein was bound to ligands, the small anion phosphate and the protein rubredoxin with the iron in the active site replaced by zinc. Comparison of these results with data for the isolated cytochrome shows that binding of ligands causes only small changes in the reduction potentials of the haems and their pairwise interactions, and also that the redox-sensitive acid,base centre responsible for the redox,Bohr effect is essentially unaffected. Although neither of the ligands tested is a physiological partner of cytochrome c3, the small changes observed for the thermodynamic properties of cytochrome c3 bound to these ligands vs. the unbound state, indicate that the thermodynamic properties measured for the isolated protein are relevant for a physiological interpretation of the role of this cytochrome in the bioenergetic metabolism of Desulfovibrio. [source] Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-TuFEBS JOURNAL, Issue 18 2004Damodara Rao The protein synthesis elongation factor, EF-Tu, is a protein that carries aminoacyl-tRNA to the A-site of the ribosome during the elongation phase of protein synthesis. In maize (Zea mays L) this protein has been implicated in heat tolerance, and it has been hypothesized that EF-Tu confers heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation and inactivation. In this study we investigated the effect of the recombinant precursor of maize EF-Tu (pre-EF-Tu) on thermal aggregation and inactivation of the heat-labile proteins, citrate synthase and malate dehydrogenase. The recombinant pre-EF-Tu was purified from Escherichia coli expressing this protein, and mass spectrometry confirmed that the isolated protein was indeed maize EF-Tu. The purified protein was capable of binding GDP (indicative of protein activity) and was stable at 45 °C, the highest temperature used in this study to test this protein for possible chaperone activity. Importantly, the recombinant maize pre-EF-Tu displayed chaperone activity. It protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation. To our knowledge, this is the first observation of chaperone activity by a plant/eukaryotic pre-EF-Tu protein. The results of this study support the hypothesis that maize EF-Tu plays a role in heat tolerance by acting as a molecular chaperone and protecting chloroplast proteins from thermal aggregation and inactivation. [source] Specific reactions of S -nitrosothiols with cysteine hydrolases: A comparative study between dimethylargininase-1 and CTP synthetasePROTEIN SCIENCE, Issue 8 2007Oliver Braun Abstract S-Transnitrosation is an important bioregulatory process whereby NO+ equivalents are transferred between S -nitrosothiols and Cys of target proteins. This reaction proceeds through a common intermediate R,S,N(O,),S,R, and it has been proposed that products different from S -nitrosothiols may be formed in protein cavities. Recently, we have reported on the formation of such a product, an N -thiosulfoximide, at the active site of the Cys hydrolase dimethylargininase-1 (DDAH-1) upon reaction with S -nitroso- l -homocysteine (HcyNO). Here we have addressed the question of whether this novel product can also be formed with the endogenously occurring S -nitrosothiols S -nitroso- l -cysteine (CysNO) and S -nitrosoglutathione (GSNO). Further, to explore the reason responsible for the unique formation of an N -thiosulfoximide in DDAH-1 we have expanded these studies to cytidine triphosphate synthetase (CTPS), which shows a similar active site architecture. ESI-MS and activity measurements showed that the bulky GSNO does not react with both enzymes. In contrast, S-nitrosylation of the active site Cys occurred in DDAH-1 with CysNO and in CTPS with CysNO and HcyNO. Although kinetic analysis indicated that these compounds act as specific irreversible inhibitors, no N -thiosulfoximide was formed. The reasons likely responsible for the absence of the N -thiosulfoximide formation are discussed using molecular models of DDAH-1 and CTPS. In tissue extracts DDAH was inhibited only by HcyNO, with an IC50 value similar to that of the isolated protein. Biological implications of these studies for the function of both enzymes are discussed. [source] Study of stationary phase metabolism via isotopomer analysis of amino acids from an isolated proteinBIOTECHNOLOGY PROGRESS, Issue 1 2010Afshan S. Shaikh Abstract Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully 13C-labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Delivery of bioactive, gel-isolated proteins into live cellsELECTROPHORESIS, Issue 9 2003Jennifer E. Taylor Abstract The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein. [source] Hydrophobins: the protein-amphiphiles of filamentous fungiFEMS MICROBIOLOGY REVIEWS, Issue 5 2005Markus B. Linder Abstract Hydrophobins are surface active proteins produced by filamentous fungi. They have a role in fungal growth as structural components and in the interaction of fungi with their environment. They have, for example, been found to be important for aerial growth, and for the attachment of fungi to solid supports. Hydrophobins also render fungal structures, such as spores, hydrophobic. The biophysical properties of the isolated proteins are remarkable, such as strong adhesion, high surface activity and the formation of various self-assembled structures. The first high resolution three dimensional structure of a hydrophobin, HFBII from Trichoderma reesei, was recently solved. In this review, the properties of hydrophobins are analyzed in light of these new data. Various application possibilities are also discussed. [source] A one-carbon modification of protein lysine associated with elevated oxidative stress in human substantia nigraJOURNAL OF NEUROCHEMISTRY, Issue 2 2006Erik Floor Abstract We describe for the first time a naturally occurring lysine modification that is converted to methyllysine by reduction with sodium borohydride. This modification is ,1.7 times as abundant in soluble proteins from human substantia nigra pars compacta as in proteins from other brain regions, possibly as a result of elevated oxidative stress in the nigra. Proteins from cultured PC12 cells exposed to oxidative stress conditions also contain elevated levels of this lysine modification. The abundance of the naturally occurring modification is roughly 0.08 nmoles/mg protein in either unstressed brain or PC12 cells. Modification levels remain stable in isolated proteins incubated for 2 h at 37°C in pH 7 buffer. We propose that the endogenous modification is the lysine Schiff base, ,- N -methylenelysine, and that lysine modifications may result from a reaction with formaldehyde in vivo. Rat brain contains ,60 nmoles/g wet weight of formaldehyde, which probably includes both free and reversibly bound forms. Adding ,35 µm HCHO to PC12 cell growth medium introduces methylenelysine modifications in cell proteins and impairs cell viability. The existence of this post-translational modification suggests new mechanisms of oxidative stress that may contribute to tissue degeneration, including loss of nigral dopamine neurons during normal aging and in Parkinson's disease. [source] | |||||||||||||