The Protein Journal最新文献

Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7–20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin α protein due to binding between the importin β-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin α from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin α. However, our data indicate that T. gondii importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.

Metamorphic, or fold-switching, proteins feature different folds that are physiologically relevant. The human chemokine XCL1 (or Lymphotactin) is a metamorphic protein that features two native states, an (alpha -beta) and an all(-beta) fold, which have similar stability at physiological condition. Here, extended molecular dynamics (MD) simulations, principal component analysis of atomic fluctuations and thermodynamic modeling based on both the configurational volume and free energy landscape, are used to obtain a detailed characterization of the conformational thermodynamics of human Lymphotactin and of one of its ancestors (as was previously obtained by genetic reconstruction). Comparison of our computational results with the available experimental data show that the MD-based thermodynamics can explain the experimentally observed variation of the conformational equilibrium between the two proteins. In particular, our computational data provide an interpretation of the thermodynamic evolution in this protein, revealing the relevance of the configurational entropy and of the shape of the free energy landscape within the essential space (i.e., the space defined by the generalized internal coordinates providing the largest, typically non-Gaussian, structural fluctuations).

Pfu DNA polymerase is one of the most preferred molecular enzymes that is isolated from the hyperthermophilic Pyrococcus furiosus and used for high-throughput DNA synthesis by the polymerase chain reaction. Therefore, an efficient Pfu DNA polymerase production method is necessary for molecular techniques. In the present study, Pfu DNA polymerase was expressed in recombinant Escherichia coli BL21(DE3) and significant parameters for the biomass production were optimized using the central composite design which is the most popular method of response surface methodology. Induction conditions including cell density prior induction (OD_600nm), post-induction temperature, IPTG concentration, and post-induction time and their interactions on biomass production were investigated. The maximum biomass production (14.1 g/L) in shake flasks was achieved using the following predicted optimal conditions: OD_600nm before induction of 0.4 and the induction at 32 °C for 7.7 h, with 0.6 mM IPTG. Optimized culture conditions were implemented to scale up experiments. 22% and 70% increase in biomass production was achieved in 3 L and 10 L bioreactors, respectively as compared to initial biomass production observed in unoptimized conditions. Similary, a 30% increase of Pfu DNA polymerase production was obtained after the optimization. The polymerase activity of the purifed Pfu DNA polymerase was assessed by PCR amplification and determined as 2.9 U/μl by comparison with commercial Pfu DNA polymerase. The findings of this study indicated that the proposed fermentation conditions will contribute to further scale‑up studies to enhance the biomass for the production of other recombinant proteins.

Due to the importance of protein-protein interactions in defence mechanism of living body, attempts were made to investigate its attributes, including, but not limited to, binding affinity, and binding region. Contemporary strategies for binding site prediction largely resort to deep learning techniques but turned out to be low precision models. As laboratory experiments for drug discovery tasks utilize this information, increased false positives devalue the computational methods. This emphasize the need to develop enhanced strategies. DeepBindPPI employs deep learning technique to predict the binding regions of proteins, particularly antigen–antibody interaction sites. The results obtained are applied in a docking environment to confirm their correctness. An integration of graph convolutional network with attention mechanism predicts interacting amino acids with improved precision. The model learns the determining factors in interaction from a general pool of proteins and is then fine-tuned using antigen–antibody data. Comparison of the proposed method with existing techniques shows that the developed model has comparable performance. The use of a separate spatial network clearly improved the precision of the proposed method from 0.4 to 0.5. An attempt to utilize the interface information for docking using the HDOCK server gives promising results, with high-quality structures appearing in the top10 ranks.

The rise of New Delhi metallo beta-lactamase (NDM) producing bacteria imposes a significant threat to the treatment of bacterial infections due to their broad spectrum against beta-lactams. The activity of metallo beta-lactamases is affected by active site residues as well as residues near the active site. Therefore, we aimed to identify the amino acid residues around the active site of NDM-4 which influence its function. To achieve that, seven substitution mutations (S191A, D192A, S213A, K216A, S217A, D223A and D225A) of NDM-4 were generated through site-directed mutagenesis. Out of these, expression of NDM-4_D192A and NDM-4_S217A in Escherichia coli cells increased the beta-lactam susceptibility as compared to NDM-4. Further, proteins were purified to assess the effect of substitution mutations on zinc content, in vitro catalytic efficiency, and stability of NDM-4. The catalytic efficiency was reduced for these mutants (D192A and S217A) towards beta-lactam substrates, while the thermal stability remained insubstantial as compared to NDM-4. However, the purified NDM-4_D192A exhibited altered zinc content. In silico studies reveal that these changes might be the outcomes of alterations in hydrogen bonding networks and substrate interactions. Taken together, we infer that the D192 and the S217 residues play a substantial role in the activity of NDM-4.

The majority of the clotting factors involved in blood coagulation pathways are serine proteases and thrombin is one of the key serine proteases involved in blood clotting. Many synthetic and chemical drugs targeting these proteases as therapeutics are known. However, they are associated with serious side effects such as bleeding, haemorrhage, edema etc. Serine protease inhibitors from plants have been suggested as one of the potential anticoagulant molecules against thrombosis. In the present work, a direct thrombin inhibitor from Moringa oleifera was isolated, purified and characterized. The homogeneity of the inhibitor is confirmed on native- PAGE. The purified inhibitor (5 µg) showed 63% thrombin inhibition at pH 7.2 at 37 °C. The IC₅₀ value of the isolated inhibitor was determined as 4.23 µg. The inhibitor on SDS-PAGE appeared as a single protein-stained band corresponding to 50 kDa thereby indicating its molecular weight as 50 kDa. Purified thrombin inhibitor (5 µg) showed 12% inhibition of trypsin, and 17% inhibition of chymotrypsin. This suggests more specificity of purified inhibitor towards thrombin. The isolated inhibitor showed a non-competitive mode of inhibition against thrombin as determined by the Dixon plot. The inhibition constant (Ki) was calculated as 4.35 × 10^–7 M. The present work reports for the first time a direct thrombin inhibitor from M. oleifera which may be further explored as an antithrombotic drug.

There still is little treatment available for amyloid diseases, despite their significant impact on individuals and the social and economic implications for society. One reason for this is that the physical nature of amyloid formation is not understood sufficiently well. Therefore, fundamental research at the molecular level remains necessary to support the development of therapeutics. A few structures of short peptides from amyloid-forming proteins have been determined. These can in principle be used as scaffolds for designing aggregation inhibitors. Attempts to this end have often used the tools of computational chemistry, in particular molecular simulation. However, few simulation studies of these peptides in the crystal state have been presented so far. Hence, to validate the capability of common force fields (AMBER19SB, CHARMM36m, and OPLS-AA/M) to yield insight into the dynamics and structural stability of amyloid peptide aggregates, we have performed molecular dynamics simulations of twelve different peptide crystals at two different temperatures. From the simulations, we evaluate the hydrogen bonding patterns, the isotropic B-factors, the change in energy, the Ramachandran plots, and the unit cell parameters and compare the results with the crystal structures. Most crystals are stable in the simulations but for all force fields there is at least one that deviates from the experimental crystal, suggesting more work is needed on these models.