Consensus protein engineering on the thermostable histone-like bacterial protein HUs significantly improves stability and DNA binding affinity

Georgoulis, Anastasios D.; Louka, Maria; Mylonas, Stratos; Stavros, Philemon; Nounesis, George; Vorgias, Constantinos E.

doi:10.1007/s00792-020-01154-4

Cited by 6 publications

(5 citation statements)

References 104 publications

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“…Ligation to perfect duplexes DNA would allow HU protein to protect them before exposure to stress, thus reducing damage. Additionally, the conserved glycine at position 15 was also observed, a residue that mediates loop flexibility and promotes the thermal stability of HU ( Kawamura et al, 1996 ; Georgoulis et al, 2020 ) ( Supplementary Figure S5A ). Likewise, the presence of the Ser-His-Asp catalytic triad was observed in ClpP sequences, a motif fully conserved in members of the ClpP family and which plays an essential role in the cleavage capacity of proteases ( Khan et al, 2022 ) ( Supplementary Figure S5B ).…”

Section: Resultsmentioning

confidence: 99%

Identification and functional analysis of novel protein-encoding sequences related to stress-resistance

Huanca-Juarez,

Nascimento-Silva,

Silva

et al. 2023

Front. Microbiol.

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Currently, industrial bioproducts are less competitive than chemically produced goods due to the shortcomings of conventional microbial hosts. Thus, is essential developing robust bacteria for improved cell tolerance to process-specific parameters. In this context, metagenomic approaches from extreme environments can provide useful biological parts to improve bacterial robustness. Here, in order to build genetic constructs that increase bacterial resistance to diverse stress conditions, we recovered novel protein-encoding sequences related to stress-resistance from metagenomic databases using an in silico approach based on Hidden-Markov-Model profiles. For this purpose, we used metagenomic shotgun sequencing data from microbial communities of extreme environments to identify genes encoding chaperones and other proteins that confer resistance to stress conditions. We identified and characterized 10 novel protein-encoding sequences related to the DNA-binding protein HU, the ATP-dependent protease ClpP, and the chaperone protein DnaJ. By expressing these genes in Escherichia coli under several stress conditions (including high temperature, acidity, oxidative and osmotic stress, and UV radiation), we identified five genes conferring resistance to at least two stress conditions when expressed in E. coli. Moreover, one of the identified HU coding-genes which was retrieved from an acidic soil metagenome increased E. coli tolerance to four different stress conditions, implying its suitability for the construction of a synthetic circuit directed to expand broad bacterial resistance.

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Section: Resultsmentioning

confidence: 99%

Identification and functional analysis of novel protein-encoding sequences related to stress-resistance

Huanca-Juarez,

Nascimento-Silva,

Silva

et al. 2023

Front. Microbiol.

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“…In order to obtain more structural information regarding the low optimal reaction temperature of xylosidase AX543, semi-rational homologous module substitution was applied in the following research. The homologous substitution strategy has been previously applied to various proteins, which can lead to the design of proteins with enhanced biological performance to meet the requirements of specific research and technological goals [ 34 , 35 ]. Previously, after fusion of the thermostabilizing domain A2 of xylanase XynA to the N-terminal region of Xyn2, the optimal temperature of the hybrid enzyme was 10 °C higher than for Xyn2, and its thermostability was also improved [ 36 ].…”

Section: Resultsmentioning

confidence: 99%

Improving the Specific Activity and Thermostability of Psychrophilic Xylosidase AX543 by Comparative Mutagenesis

Pan

Liu

Zhang

et al. 2022

Foods

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Improving the specific activity and thermostability of psychrophilic xylosidase is important for improving its enzymatic performance and promoting its industrial application. Herein, a psychrophilic xylosidase AX543 exhibited activity in the temperature range between 0 and 35 °C, with optimum activity at 20 °C, which is lower than that of other reported psychrophilic xylosidases. The thermostability, specific activity, and catalytic efficiency of the site-directed variants G110S, Q201R, and L2 were significantly enhanced, without affecting the optimal reaction temperature. Comparative protein structural analysis and molecular dynamics simulation indicated that these improvements might be the result of the increased hydrogen bonds interaction and improved structural rigidity. Furthermore, homologous module substitution with four segments demonstrated that the psychrophilic characteristics of AX543 are the results of the whole protein structure, and the C-terminal segment A4 appears to be more essential in determining psychrophilic characteristics, exhibiting potentiality to produce more psychrophilic xylosidases. This study provides valuable structural information on psychrophilic xylosidases and also offers attractive modification strategies to modify catalytic activity, thermostability, and optimal reaction temperature.

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“…Protein engineering studies often seek improved stabilities. Several evolutionary‐based strategies, such as back‐to‐consensus mutations 53 , 54 , 55 and ancestral protein reconstruction, 21 , 56 , 57 have been well used to achieve protein stabilization. Also, directed evolution techniques have shown great promise in stabilizing proteins.…”

Section: Discussionmentioning

confidence: 99%

Active site center redesign increases protein stability preserving catalysis in thioredoxin

2022

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The stabilization of natural proteins is a long‐standing desired goal in protein engineering. Optimizing the hydrophobicity of the protein core often results in extensive stability enhancements. However, the presence of totally or partially buried catalytic charged residues, essential for protein function, has limited the applicability of this strategy. Here, focusing on the thioredoxin, we aimed to augment protein stability by removing buried charged residues in the active site without loss of catalytic activity. To this end, we performed a charged‐to‐hydrophobic substitution of a buried and functional group, resulting in a significant stability increase yet abolishing catalytic activity. Then, to simulate the catalytic role of the buried ionizable group, we designed a combinatorial library of variants targeting a set of seven surface residues adjacent to the active site. Notably, more than 50% of the library variants restored, to some extent, the catalytic activity. The combination of experimental study of 2% of the library with the prediction of the whole mutational space by partial least squares regression revealed that a single point mutation at the protein surface is sufficient to fully restore the catalytic activity without thermostability cost. As a result, we engineered one of the highest thermal stabilities reported for a protein with a natural occurring fold (137°C). Further, our hyperstable variant preserves the catalytic activity both in vitro and in vivo.

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Consensus protein engineering on the thermostable histone-like bacterial protein HUs significantly improves stability and DNA binding affinity

Cited by 6 publications

References 104 publications

Identification and functional analysis of novel protein-encoding sequences related to stress-resistance

Identification and functional analysis of novel protein-encoding sequences related to stress-resistance

Improving the Specific Activity and Thermostability of Psychrophilic Xylosidase AX543 by Comparative Mutagenesis

Active site center redesign increases protein stability preserving catalysis in thioredoxin

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