Biochimica et biophysica acta. Proteins and proteomics最新文献

Implementing hyperthermostable carbonic anhydrases into CO₂ capture and storage technologies in order to increase the rate of CO₂ absorption from the industrial flue gases is of great importance from technical and economical points of view. The present study employed a combination of in silico tools to further improve thermostability of a known thermostable carbonic anhydrase from Sulfurihydrogenibium yellowstonense. Experimental results showed that our rationally engineered K100G mutant not only retained the overall structure and catalytic efficiency but also showed a 3 °C increase in the melting temperature and a two-fold improvement in the enzyme half-life at 85 °C. Based on the molecular dynamics simulation results, rearrangement of salt bridges and hydrogen interactions network causes a reduction in local flexibility of the K100G variant. In conclusion, our study demonstrated that thermostability can be improved through imposing local structural rigidity by engineering a single-point mutation on the surface of the enzyme.

Cellulases from anaerobic fungi are enzymes less-studied biochemically and structurally than cellulases from bacteria and aerobic fungi. Currently, only thirteen GH5 cellulases from anaerobic fungi were biochemically characterized and two crystal structures were reported. In this context, here, we report the functional and biophysical characterization of a novel multi-modular cellulosomal GH5 endoglucanase from the anaerobic gut fungus Piromyces finnis (named here PfGH5). Multiple sequences alignments indicate that PfGH5 is composed of a GH5 catalytic domain and a CBM1 carbohydrate-binding module connected through a CBM10 dockerin module. Our results showed that PfGH5 is an endoglucanase from anaerobic fungus with a large spectrum of activity. PfGH5 exhibited preference for hydrolysis of oat β-glucan, followed by galactomannan, carboxymethyl cellulose, mannan, lichenan and barley β-glucan, therefore displaying multi-functionality. For oat β-glucan, PfGH5 reaches its optimum enzymatic activity at 40 °C and pH 5.5, with K_m of 7.1 μM. Ion exchange chromatography analyzes revealed the production of oligosaccharides with a wide degree of polymerization indicated that PfGH5 has endoglucanase activity. The ability to bind and cleave different types of carbohydrates evidence the potential of PfGH5 for use in biotechnology and provide a useful basis for future investigation and application of new anaerobic fungi enzymes.

Human α-, β-, and γ-synuclein (syn) are natively unfolded proteins present in the brain. Deposition of aggregated α-syn in Lewy bodies is associated with Parkinson's disease (PD) and γ-syn is known to be involved in both neurodegeneration and breast cancer. At physiological pH, while α-syn has the highest propensity for fibrillation followed by γ-syn, β-syn does not form any fibrils. Fibril formation in these proteins could be modulated by protein structure stabilizing osmolytes such as trehalose which has an exceptional stabilizing effect for globular proteins. We present a comprehensive study of the effect of trehalose on the conformation, aggregation, and fibril morphology of α-, β-, and γ-syn proteins. Rather than stabilizing the intrinsically disordered state of the synucleins, trehalose accelerates the rate of fibril formation by forming aggregation-competent partially folded intermediate structures. Fibril morphologies are also strongly dependent on the concentration of trehalose with ≤ 0.4M favoring the formation of mature fibrils in α-, and γ-syn with no effect on the fibrillation of β-syn. At ≥ 0.8M, trehalose promotes the formation of smaller aggregates that are more cytotoxic. Live cell imaging of preformed aggregates of a labeled A90C α-syn shows their rapid internalization into neural cells which could be useful in reducing the load of aggregated species of α-syn. The findings throw light on the differential effect of trehalose on the conformation and aggregation of disordered synuclein proteins with respect to globular proteins and could help in understanding the effect of osmolytes on intrinsically disordered proteins under cellular stress conditions.

Fe and Zn ions are essential enzymatic cofactors across all domains of life. Fe is an electron donor/acceptor in redox enzymes, while Zn is typically a structural element or catalytic component in hydrolases. Interestingly, the presence of Zn in oxidoreductases and Fe in hydrolases challenge this apparent functional dichotomy. In hydrolases, Fe either substitutes for Zn or specifically catalyzes certain reactions. On the other hand, Zn can replace divalent Fe and substitute for more complex Fe assemblies, known as Fe-S clusters. Although many zinc-binding proteins interchangeably harbor Zn and Fe-S clusters, these cofactors are only sometimes functional proxies.

Crystallins are the major soluble lens proteins, and α-crystallin, the most important protective protein of the eye lens, has two subunits (αA and αB) with chaperone activity. αB-crystallin (αB-Cry) with a relatively wide tissue distribution has an innate ability to interact effectively with the misfolded proteins, preventing their aggregation. Melatonin and serotonin have also been identified in relatively high concentrations in the lenticular tissues. This study investigated the effect of these naturally occurring compounds and medications on the structure, oligomerization, aggregation, and chaperone-like activity of human αB-Cry. Various spectroscopic methods, dynamic light scattering (DLS), differential scanning calorimetry (DSC), and molecular docking have been used for this purpose. Based on our results, melatonin indicates an inhibitory effect on the aggregation of human αB-Cry without altering its chaperone-like activity. However, serotonin decreases αB-Cry oligomeric size distribution by creating hydrogen bonds, decreases its chaperone-like activity, and at high concentrations increases protein aggregation.

DNA replication stops when chemical or physical damage occurs to the DNA. Repairing genomic DNA and reloading the replication helicase are crucial steps for restarting DNA replication. The Escherichia coli primosome is a complex of proteins and DNA responsible for reloading the replication helicase DnaB. DnaT, a protein found in the primosome complex, contains two functional domains. The C-terminal domain (89–179) forms an oligomeric complex with single-stranded DNA. Although the N-terminal domain (1–88) forms an oligomer, the specific residues responsible for this oligomeric structure have not yet been identified.

In this study, we proposed that the N-terminal domain of DnaT has a dimeric antitoxin structure based on its primary sequence. Based on the proposed model, we confirmed the site of oligomerization in the N-terminal domain of DnaT through site-directed mutagenesis. The molecular masses and thermodynamic stabilities of the site-directed mutants located at the dimer interface, namely Phe42, Tyr43, Leu50, Leu53, and Leu54, were found to be lower than those of the wild-type. Moreover, we observed a decrease in the molecular masses of the V10S and F35S mutants compared to the wild-type DnaT. NMR analysis of the V10S mutant revealed that the secondary structure of the N-terminal domain of DnaT was consistent with the proposed model. Additionally, we have demonstrated that the stability of the oligomer formed by the N-terminal domain of DnaT is crucial for its function. Based on these findings, we propose that the DnaT oligomer plays a role in replication restart in Escherichia coli.

Sea anemone venoms are complex mixtures of biologically active compounds, including disulfide-rich peptides, some of which have found applications as research tools, and others as therapeutic leads. Our recent transcriptomic and proteomic studies of the Australian sea anemone Telmatactis stephensoni identified a transcript for a peptide designated Tst2. Tst2 is a 38-residue peptide showing sequence similarity to peptide toxins known to interact with a range of ion channels (Na_V, TRPV1, K_V and Ca_V). Recombinant Tst2 (rTst2, which contains an additional Gly at the N-terminus) was produced by periplasmic expression in Escherichia coli, enabling the production of both unlabelled and uniformly ¹³C,¹⁵N–labelled peptide for functional assays and structural studies. The LC-MS profile of the recombinant Tst2 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds from its six cysteine residues. The solution structure of rTst2 was determined using multidimensional NMR spectroscopy and revealed that rTst2 adopts an inhibitor cystine knot (ICK) structure. rTst2 was screened using various functional assays, including patch–clamp electrophysiological and cytotoxicity assays. rTst2 was inactive against voltage-gated sodium channels (Na_V) and the human voltage-gated proton (hHv1) channel. rTst2 also did not possess cytotoxic activity when assessed against Drosophila melanogaster flies. However, the recombinant peptide at 100 nM showed >50% inhibition of the transient receptor potential subfamily V member 1 (TRPV1) and slight (∼10%) inhibition of transient receptor potential subfamily A member 1 (TRPA1). Tst2 is thus a novel ICK inhibitor of the TRPV1 channel.

The second most prevalent neurodegenerative disease, Parkinson's disease (PD), is caused by the accumulation and deposition of fibrillar aggregates of the α-Syn into the Lewy bodies. To create a potent pharmacological candidate to destabilize the preformed α-Syn fibril, it is important to understand the precise molecular mechanism underlying the destabilization of the α-Syn fibril. Through molecular dynamics simulations and experiments, we have examined the molecular mechanisms causing the destabilization and suppression of a newly discovered α-Syn fibril with a Greek-key-like shape and an aggregation prone state (APS) of α-Syn in the presence and absence of various Flvs. According to MD simulation and experimental evidence, morin, quercetin, and myricetin are the Flvs, most capable of destabilizing the fibrils and converting them into amorphous aggregates. Compared to galangin and kaempferol, they have more hydroxyl groups and form more hydrogen bonds with fibrils.The processes by which morin and myricetin prevent new fibril production from APS and destabilize the fibrils are different. According to linear interaction energy analysis, van der Waals interaction predominates with morin, and electrostatic interaction dominates with myricetin. Our MD simulation and experimental findings provide mechanistic insights into how Flvs destabilize α-Syn fibrils and change their morphology, opening the door to developing structure-based drugs for treating Parkinson's disease.

Tauopathies and synucleinopathies are characterized by the aggregation of Tau and α-synuclein (AS) into amyloid structures, respectively. Individuals with these neuropathies have an elevated risk of developing subsequent neurodegenerative or comorbid disorders. Intriguingly, post-mortem brain examinations have revealed co-localization of Tau and AS aggregates, suggesting a synergistic pathological relationship with an adverse prognosis. The role of liquid-liquid phase separation (LLPS) in the development of neurodegenerative diseases is currently receiving significant attention, as it can contribute to the aggregation and co-deposition of amyloidogenic proteins. In this study, we investigated the phase separation behavior of Tau and AS under various insults, some of which are implicated in disease progression. Our findings demonstrate the formation of heterotypic droplets composed of Tau and AS at physiologically relevant mole ratios that mimic neurons' soma and terminal buttons. Importantly, these heterotypic droplets exhibit increased resistance to electrostatic screening compared to homotypic condensates. Moreover, we observed that biologically relevant biomolecules, known to be dysregulated in disease, exert different effects on these droplets. Additionally, we provide evidence that phase separation itself influences the amyloid aggregation of Tau and AS, underscoring the significance of this process in the development of aggregopathies.

Over the last 40 years nuclear magnetic resonance (NMR) spectroscopy has established itself as one of the most versatile techniques for the characterization of biomolecules, especially proteins. Given the molecular size limitations of NMR together with recent advances in cryo-electron microscopy and artificial intelligence-assisted protein structure prediction, the bright future of NMR in structural biology has been put into question. In this mini review we argue the contrary. We discuss the unique opportunities solution NMR offers to the protein chemist that distinguish it from all other experimental or computational methods, and how it can benefit from machine learning.