62 research outputs found
Homology-derived three-dimensional structure prediction of Candida cylindracea lipase
No full textWe propose a structural model of Candida cylindracea lipase (CCL) based on the reported X-ray structure of the highly homologous Geotrichum candidum lipase (GCL). The network of interactions around the active site, the salt and disulfide bridge pattern is conserved in the proposed structure. Functional, structural and evolutionary aspects of the peculiar usage of CTG codons by C. cylindracea ATCC 14830 are discussed. © 1992
Domain Interactions and Close Vicinity of trans Reentrant Loops in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae (vol 49, pg 4509, 2010)
No full textBacillus subtilis SpoIIIJ and YqjG Function in Membrane Protein Biogenesis
No full textIn all domains of life Oxa1p-like proteins are involved in membrane protein biogenesis. Bacillus subtilis, a model organism for gram-positive bacteria, contains two Oxa1p homologs: SpoIIIJ and YqjG. These molecules appear to be mutually exchangeable, although SpoIIIJ is specifically required for spore formation. SpoIIIJ and YqjG have been implicated in a posttranslocational stage of protein secretion. Here we show that the expression of either spoIIIJ or yqjG functionally compensates for the defects in membrane insertion due to YidC depletion in Escherichia coli. Both SpoIIIJ and YqjG complement the function of YidC in SecYEG-dependent and -independent membrane insertion of subunits of the cytochrome o oxidase and F1Fo ATP synthase complexes. Furthermore, SpoIIIJ and YqjG facilitate membrane insertion of F1Fo ATP synthase subunit c from both E. coli and B. subtilis into inner membrane vesicles of E. coli. When isolated from B. subtilis cells, SpoIIIJ and YqjG were found to be associated with the entire F1Fo ATP synthase complex, suggesting that they have a role late in the membrane assembly process. These data demonstrate that the Bacillus Oxa1p homologs have a role in membrane protein biogenesis rather than in protein secretion.
Structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli using 5-FTrp fluorescence spectroscopy
No full textThe mannitol transporter EIImtl from Escherichia coli is responsible for the uptake of mannitol over the inner membrane and its concomitant phosphorylation. EIImtl is functional as a dimer and its membrane-embedded C domain, IICmtl, harbors one high affinity mannitol binding site. To characterize this domain in more detail the microenvironments of thirteen residue positions were explored by 5-fluorotryptophan (5-FTrp) fluorescence spectroscopy. Because of the simpler photophysics of 5-FTrp compared to Trp, one can distinguish between the two 5-FTrp probes present in dimeric IICmtl. At many labeled positions, the microenvironment of the 5-FTrps in the two protomers differs. Spectroscopic properties of three mutants labeled at positions 198, 251, and 260 show that two conserved motifs (Asn194-His195 and Gly254-Ile255-His256-Glu257) are located in well-structured parts of IICmtl. Mannitol binding has a large impact on the structure around position 198, while only minor changes are induced at positions 251 and 260. Phosphorylation of the cytoplasmic B domain of EIImtl is sensed by 5-FTrp at positions 30, 42, 251 and 260. We conclude that many parts of the IICmtl structure are involved in the sugar translocation. The structure of EIImtl, as investigated in this work, differs from the recently solved structure of a IIC protein transporting diacetylchitobiose, ChbC, and also belonging to the glucose superfamily of EII sugar transporters. In EIImtl, the sugar binding site is more close to the periplasmic face and the structure of the 2 protomers in the dimer is different, while both protomers in the ChbC dimer are essentially the same.
Purification, Crystallisation and Preliminary X-ray Analysis of Penicillin Binding Protein 4 from Escherichia coli, a Protein Related to Class A β-Lactamases
No full textCrystals of the penicillin binding protein 4 (PBP4) from Escherichia coli have been obtained at 37°C from liquid to liquid diffusion experiments in capillaries. PBP4 was dissolved in a 1.0 M ammonium sulphate solution, buffered at pH 7.2, to a concentration of 5 mg/ml, and was layered on top of a 1.6 to 2.2 M ammonium sulphate solution. Crystals appeared within four to six weeks. They belong to space group C222 with cell dimensions a = 68.5 Å, b = 100.5 Å and c = 137.0 Å, and diffract to at least 2.8 Å resolution. There is one molecule with a molecule mass of 49,568 Da in the asymmetric unit.
Enantioselective Artificial Metalloenzymes by Creation of a Novel Active Site at the Protein Dimer Interface
No full textArtificial metalloenzymes are generated by forming a novel active site on the dimer interface of the transcription factor LmrR. Two copper centers are incorporated by binding to ligands in each half of the dimer. With this system up to 97 % ee was obtained in the benchmark CuII catalyzed Diels–Alder reaction.
Proteolysin, a Novel Highly Thermostable and Cosolvent-Compatible Protease from the Thermophilic Bacterium Coprothermobacter proteolyticus
No full textThrough genome mining, we identified a gene encoding a putative serine protease of the thermitase subgroup of subtilases (EC 3.4.21.66) in the thermophilic bacterium Coprothermobacter proteolyticus. The gene was functionally expressed in Escherichia coli, and the enzyme, which we called proteolysin, was purified to near homogeneity from crude cell lysate by a single heat treatment step. Proteolysin has a broad pH tolerance and is active at temperatures of up to 80°C. In addition, the enzyme shows good activity and stability in the presence of organic solvents, detergents, and dithiothreitol, and it remains active in 6 M guanidinium hydrochloride. Based on its stability and activity profile, proteolysin can be an excellent candidate for applications where resistance to harsh process conditions is required.
A proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers
No full textInteractions between hematopoietic stem cells and their niche are mediated by proteins within the plasma membrane (PM) and changes in these interactions might alter hematopoietic stem cell fate and ultimately result in acute myeloid leukemia (AML). Here, using nano-LC/MS/MS, we set out to analyze the PM profile of two leukemia patient samples. We identified 867 and 610 unique CD34(+) PM (-associated) proteins in these AML samples respectively, including previously described proteins such as CD47, CD44, CD135, CD96, and ITGA5, but also novel ones like CD82, CD97, CD99, PTH2R, ESAM, MET, and ITGA6. Further validation by flow cytometry and functional studies indicated that long-term self-renewing leukemic stem cells reside within the CD34(+)/ITGA6(+) fraction, at least in a subset of AML cases. Furthermore, we combined proteomics with transcriptomics approaches using a large panel of AML CD34(+) (n = 60) and normal bone marrow CD34(+) (n = 40) samples. Thus, we identified eight subgroups of AML patients based on their specific PM expression profile. GSEA analysis revealed that these eight subgroups are enriched for specific cellular processes
Biomarker Discovery In Chronic Obstructive Pulmonary Disease (COPD) Using Epithelial Lining Fluid: A Proteomic Approach
No full textCrystal structure of the catalytic domain of human phenylalanine hydroxylase reveals the structural basis for phenylketonuria
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