TKSA-MC: A Web Server for rational mutation through the optimization of protein charge interactions

Contessoto, Vinícius G.; Oliveira, Vinícius Martins de; Fernandes, Bruno R.; Slade, Gabriel Gouvêa; Leite, Vitor Barbanti Pereira

doi:10.1101/221556

Cited by 7 publications

(9 citation statements)

References 28 publications

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“…In order to probe the electrostatics of this charge-reversal stabilising mutation, Tanford-Kirkwood electrostatic calculations have been performed using the TKSA-MC server. A significant stabilisation of E11R is observed across a wide pH range from 2 to 12, relative to the WT (53). Electrostatic potential surface maps generated using the UCSF Chimera package show the appearance of a substantial positive - potential surface patch in the loop from residues 8 - 15 harbouring the E11R mutation ( Additional file 1: Figure S1I-J) (54).…”

Section: Discussionmentioning

confidence: 99%

Mechanistic insights into global suppressors of protein folding defects

Chattopadhyay

Bhowmick

Manjunath

et al. 2021

Preprint

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Most amino acid substitutions in a protein either lead to partial loss of function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss of function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined five global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. The suppressors both alone, and in conjunction with inactive mutants, stabilise the protein both thermodynamically and kinetically in-vitro, predominantly through acceleration of the refolding rate parameters. When coupled to inactive mutants they promote increased in-vivo solubilities as well as regain-of-function phenotypes. Our study also demonstrates that the global suppressor approach can be used to consistently stabilise wild-type proteins, including for downstream translational applications.

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Section: Discussionmentioning

confidence: 99%

Mechanistic insights into global suppressors of protein folding defects

Chattopadhyay

Bhowmick

Manjunath

et al. 2021

Preprint

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“…The TKSA-MC is a Web server developed by our research group and is used to calculate the electrostatic interactions between all ionizable residues of globular proteins via the Tanford−Kirkwood method, which includes a solvent accessibility correction. 41 The all-atoms coordinate of the native structure of the protein is used as an input of this program, and the Metropolis Monte Carlo algorithm samples the protonation states of ionizable atoms. The server provides quantitative information about the importance of each residue to protein thermal stability, and it has been a robust tool to find rational mutations that improve thermal stability in different proteins.…”

Section: Methodsmentioning

confidence: 99%

“…36,37 However, in this work, we propose a new mutation capable of optimizing the thermostability of this Bs-CspB variant and present evidence of the effect that electrostatic rearrangement has on the folding pathway and stability of this protein in its wild and variant forms. We use the TKSA-MC server 41 to seek rational mutations capable of optimizing the thermostability of the folded protein. Also, a new approach to simulate the folding at constant pH using a Monte Carlo (MC) algorithm is introducedCpHMCso we can analyze the effect of mutations on this folding process.…”

Section: Introductionmentioning

confidence: 99%

pH and Charged Mutations Modulate Cold Shock Protein Folding and Stability: A Constant pH Monte Carlo Study

Oliveira

Caetano

Silva

et al. 2019

J. Chem. Theory Comput.

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The folding and stability of proteins is a fundamental problem in several research fields. In the present paper, we have used different computational approaches to study the effects caused by changes in pH and for charged mutations in cold shock proteins from Bacillus subtilis (Bs-CspB). First, we have investigated the contribution of each ionizable residue for these proteins to their thermal stability using the TKSA-MC, a Web server for rational mutation via optimizing the protein charge interactions. Based on these results, we have proposed a new mutation in an already optimized Bs-CspB variant. We have evaluated the effects of this new mutation in the folding energy landscape using structure-based models in Monte Carlo simulation at constant pH, SBM-CpHMC. Our results using this approach have indicated that the charge rearrangements already in the unfolded state are critical to the thermal stability of Bs-CspB. Furthermore, the conjunction of these simplified methods was able not only to predict stabilizing mutations in different pHs but also to provide essential information about their effects in each stage of protein folding.

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“…However, they are often underestimated or poorly predicted during enzyme engineering. TKSA-MC [18] and pStab [19] tools tackle this issue by assessing unfavorable electrostatic interactions and identifying charged hot-spot residues for mutagenesis.…”

Section: Engineering Protein Stabilitymentioning

confidence: 99%

Web-based tools for computational enzyme design

Marques

Planas-Iglesias

Damborský

2021

Current Opinion in Structural Biology

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Enzymes are on high demand for very diverse biotechnological applications. However, natural biocatalysts often need to be engineered for fine-tuning their properties towards the end applications, such as the activity, selectivity, stability to temperature or co-solvents, and solubility. Computational methods are increasingly used in this task, providing predictions that narrow down the space of possible mutations significantly and can enormously reduce the experimental burden. Many computational tools are available as web-based platforms, making them accessible to non-expert users.These platforms are typically user-friendly, contain walk-throughs, and do not require deep expertise and installations. Here we describe some of the most recent outstanding web-tools for enzyme engineering and formulate future perspectives in this field.

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TKSA-MC: A Web Server for rational mutation through the optimization of protein charge interactions

Cited by 7 publications

References 28 publications

Mechanistic insights into global suppressors of protein folding defects

Mechanistic insights into global suppressors of protein folding defects

pH and Charged Mutations Modulate Cold Shock Protein Folding and Stability: A Constant pH Monte Carlo Study

Web-based tools for computational enzyme design

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