The Protein Journal最新文献

In this contribution I describe my personal memories of my work with Herman Berendsen and the Biophysical chemistry group in Groningen in the period of years from 1967 to 1975.

Due to its ability to reversibly bind O₂, alongside a relatively low redox reactivity and a limited cytotoxicity, the oxygen-carrying protein hemerythrin has been considered as an alternative to hemoglobin in preparing blood substitutes. In order to increase the hydrodynamic volume and lower antigenicity, two site-directed variants, H82C and K92C, were engineered that contained a single cysteine residue on the surface of each hemerythrin octamer for the specific attachment of polyethylene glycol (PEG). A sulfhydryl-reactive PEGylation reagent with a 51.9 Å spacer arm was used for selective cysteine derivatization. The mutants were characterized by UV-vis spectroscopy, size-exclusion chromatography, oxygen affinity, and autooxidation rate measurements. The H82C variant showed altered oligomeric behavior compared to the wild-type and was unstable in the met form. The PEGylated K92C variant is reasonably stable, displays an oxygen affinity similar to that of the wild-type, and shows an increased rate of autoxidation; the latter disadvantage may be counteracted by further chemical modifications.

The complete enzymatic degradation of lignocellulosic biomass requires the cooperative action of cellulosic, hemicellulosic, and lignolytic enzymes such as cellulase, xylanase, laccase, galactosidase, and arabinofuranosidase. Arabinofuranosidases (E.C 3.2.1.55), which belong to the glycoside hydrolase family of enzymes, hydrolyze the 1,3- and 1,5-α-arabinosyl bonds in L-arabinose- containing molecules. L-arabinoses are present in hemicellulosic part of lignocellulosic biomass. Arabinofuranosidases also play an important role in the complete hydrolysis of arabinoxylans. Analysis of the genome project and CAZY database revealed two putative arabinofuranosidase genes in the A. acidocaldarius genome. The aim of the study was cloning, heterologous expression, purification and biochemical characterization of the arabinofuranosidase enzyme encoded in A. acidocaldarius genome. For this purpose, the AbfA gene of the arabinofuranosidase protein was cloned into the pQE-40 vector, heterologously expressed in E. coli BL21 GOLD (DE3) and successfully purified using His-Tag. Biochemical characterization of the purified enzyme revealed that A. acidocaldarius arabinofuranosidase exhibited activity over a wide pH and temperature range with optimum activity at 45 ºC and pH 6.5 in phosphate buffer towards 4-nitrophenyl-α-L-arabinofuranoside as the substrate. In addition, the enzyme is highly stable over wide range of temperature and maintaining 60% of its activity after 90 min of incubation at 80 ºC. Through the bioinformatics studies, the homology model of A. acidocaldarius arabinofuranosidase was generated and the substrate binding site and residues located in this site were identified. Further molecular docking analysis revealed that the substrate located in the catalytically active pose and, residues N174, E175, and E294 have direct interaction with 4-nitrophenyl-α-L-arabinofuranoside. Moreover, based on phylogenetic analysis, A. acidocaldarius arabinofuranosidase exists in the sub-group of intracellular arabinofuranosidases, and G. stearothermophilus and B.subtilis arabinofuranosidases are close relatives of A. acidocaldarius arabinofuranosidase. This is the first study to report the gene cloning, recombinant expression and biochemical and bioinformatic characterization of an auxiliary GH51 arabinofuranosidase from an acidothermophilic bacterium A. acidocaldarius.

Quorum sensing (QS) is the process by which microorganisms employ chemicals called autoinducers (AIs) to communicate with their population. The QS mechanism generally controls the expression of the virulence related genes in bacteria. N-acyl homoserine lactones (AHLs) are the most widespread QS molecules. Due to their diverse AHL-lactonase activities, Bacillus species make particularly suitable candidates for procedures such as demolition of pathogenic bacterial QS signals and bioremediation of β-lactam antibiotics from contaminated environments. In this study, seven Bacillus strains with Quorum quenching (QQ) activity were isolated using an enrichment medium supplemented with Penicillin G (PenG). The AHL-lactonase encoding gene (aiiA) was amplified by PCR and sequenced. Amino acid sequences underwent multiple sequence alignment. Docking studies were carried out with both C6HSL and PenG ligand using AutoDock tools. The aiiA amino acid sequences of the isolates were found to be well conserved. Furthermore, amino acid sequence alignment revealed that 74.9% of amino acid sequences were conserved in the genus Bacillus. Docking of the C6HSL to wild type (3DHA) and H97D variant reduced the docking score by only 0.1 kcal/mol for the mutated protein. When PenG docked with a higher (1.5 kcal/mol) score as a ligand to wild-type and mutant receptors, the docking score for the mutated protein likewise decreased by 0.1 kcal/mol. This research contributed to the diversification of organisms with QQ activity and beta-lactam antibiotic resistance. It also clarified the binding score of the PenG ligand to the Bacillus AHL lactonase molecule for the first time.

In many bacteria, the High Temperature requirement A (HtrA) protein functions as a chaperone and protease. HtrA is an important factor in stress tolerance and plays a significant role in the virulence of several pathogenic bacteria. Camostat, gabexate and nafamostat mesylates are serine protease inhibitors and have recently shown a great impact in the inhibition studies of SARS-CoV2. In this study, the inhibition of Listeria monocytogenes HtrA (LmHtrA) protease activity was analysed using these three inhibitors. The cleavage assay, using human fibrinogen and casein as substrates, revealed that the three inhibitors effectively inhibit the protease activity of LmHtrA. The agar plate assay and spectrophotometric analysis concluded that the inhibition of nafamostat (IC₅₀ value of 6.6 ± 0.4 µM) is more effective compared to the other two inhibitors. Previous studies revealed that at the active site of the protease, these inhibitors are hydrolysed and one of the hydrolysates is covalently bound to the active site serine. To understand the mode of binding of these inhibitors at the active site of LmHtrA, docking of the inhibitors followed by molecular dynamics simulations were carried out. Analysis of the LmHtrA-inhibitor complex structures revealed that the covalently bound inhibitor is unable to occupy the S1 pocket of the LmHtrA which is in contrast to the previously determined camostat and nafamostat complex structures. This observation provides the first glimpse of the substrate specificity of LmHtrA which is not known. The obtained results also suggest that the development of novel inhibitors of LmHtrA and its homologs with active site architecture similar to LmHtrA can be pursued with suitable modification of these inhibitors. To date, only a very few studies have been carried out on identifying the inhibitors of HtrA proteolytic activity.

Analysing protein conformational ensembles whether from molecular dynamics (MD) simulation or other sources for functionally relevant conformational changes can be very challenging. In the nineteen nineties dimensional reduction methods were developed primarily for analysing MD trajectories to determine dominant motions with the aim of understanding their relationship to function. Coarse-graining methods were also developed so the conformational change between two structures could be described in terms of the relative motion of a small number of quasi-rigid regions rather than in terms of a large number of atoms. When these methods are combined, they can characterize the large-scale motions inherent in a conformational ensemble providing insight into possible functional mechanism. The dimensional reduction methods first applied to protein conformational ensembles were referred to as Quasi-Harmonic Analysis, Principal Component Analysis and Essential Dynamics Analysis. A retrospective on the origin of these methods is presented, the relationships between them explained, and more recent developments reviewed.

This paper describes the scientific work of Prof. Dr. Herman Berendsen on NMR spectroscopy and includes some personal notes. Since 1975, Berendsen and the author were colleagues in the Physical Chemistry group in Groningen for a period of 12 years.

This letter gives a description of my interactions with Herman over more than 30 years. His teaching, his tremendous insight in subjects newly presented to him, his generousness, his playful interactions with colleagues and students, the freedom in research direction that he allowed and also expected from his PhD students.

Proteins can be oriented in the gas phase using strong electric fields, which brings advantages for structure determination using X-ray free electron lasers. Both the vacuum conditions and the electric-field exposure risk damaging the protein structures. Here, we employ molecular dynamics simulations to rehydrate and relax vacuum and electric-field exposed proteins in aqueous solution, which simulates a refinement of structure models derived from oriented gas-phase proteins. We find that the impact of the strong electric fields on the protein structures is of minor importance after rehydration, compared to that of vacuum exposure and ionization in electrospraying. The structures did not fully relax back to their native structure in solution on the simulated timescales of 200 ns, but they recover several features, including native-like intra-protein contacts, which suggests that the structures remain in a state from which the fully native structure is accessible. Our findings imply that the electric fields used in native mass spectrometry are well below a destructive level, and suggest that structures inferred from X-ray diffraction from gas-phase proteins are relevant for solution and in vivo conditions, at least after in silico rehydration.

Xanthorhodopsin (XR) from Salinibacter ruber is a light-driven proton pump containing retinal and a light-harvesting carotenoid antenna salinixanthin. Previous structure-functional studies of XR were conducted using a protein isolated from the native host only due to the absence of heterologous expression in Escherichia coli. In this paper, we describe cell-free synthesis and incorporation in lipid–protein nanodiscs of the recombinant XR that demonstrated its principal compatibility with E. coli biosynthetic machinery. To produce XR in E. coli, three C-terminal deletion variants of this protein were constructed. In contrast to the full-length XR, their expression resulted in efficient synthesis in E. coli cells. However, cells producing recombinant XR variants bound retinal only upon growth in minimal medium, not in the rich one. The XR3 variant with deletion of ten C-terminal amino acid residues was obtained and characterized. Its absorption spectrum and photocycle kinetics were close to those reported for XR isolated from S. ruber membranes and bleached from salinixanthin. We have also constructed the first mutants of XR, H62M and D96N, and examined their properties.