Engineering proteins for thermostability through rigidifying flexible sites

Yu, Haoran; Huang, He

doi:10.1016/j.biotechadv.2013.10.012

Cited by 206 publications

(150 citation statements)

References 88 publications

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“…The fact that H489P mutation successfully enhances the thermostability of luciferase without changing its activity and other structure properties indicates that rigidifying flexible region 487-495 will be a promising approach to enhance thermal stability of P. pyralis luciferase. Based on our knowledge, Fragment 487-495 can be further rigidified through methods including ISM (Iterative Saturation Mutagenesis), introduction of salt bridge or disulfide bonds and Rosetta Design [7].…”

Section: Discussionmentioning

confidence: 99%

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The role of proline substitutions within flexible regions on thermostability of luciferase

Zhao

Guo

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Section: Discussionmentioning

confidence: 99%

“…Recently, flexibility has been proved to be an effective indicator to find out weak spots. A protein engineering approach named RFS (rigidify flexible sites) has been widely used to enhance thermostability of proteins, which contains two steps: predict flexible sites and rigidify these sites [7].…”

Section: Introductionmentioning

confidence: 99%

The role of proline substitutions within flexible regions on thermostability of luciferase

Zhao

Guo

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Self Cite

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“…Beneficial mutations that lead to increased thermostability or decreased thermoactivity, improved pH-optimum or solubility can be involved in many different mechanisms determined by the laws of physics and chemistry including solvent interactions, structural support, and electrostatic balance. 59,94,95,96,97,98,99,100 Consequently, the search for variants with improved function is best treated as a combinatorial optimization problem, in which a number of parameters must be optimised simultaneously to achieve a successful outcome imitating the nature's manner.…”

Section: Changing Specificitymentioning

confidence: 99%

Genetically modified proteins: functional improvement and chimeragenesis

et al. 2015

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This review focuses on the emerging role of site-specific mutagenesis and chimeragenesis for the functional improvement of proteins in areas where traditional protein engineering methods have been extensively used and practically exhausted. The novel path for the creation of the novel proteins has been created on the farther development of the new structure and sequence optimization algorithms for generating and designing the accurate structure models in result of x-ray crystallography studies of a lot of proteins and their mutant forms. Artificial genetic modifications aim to expand nature's repertoire of biomolecules. One of the most exciting potential results of mutagenesis or chimeragenesis finding could be design of effective diagnostics, bio-therapeutics and biocatalysts. A sampling of recent examples is listed below for the in vivo and in vitro genetically improvement of various binding protein and enzyme functions, with references for more in-depth study provided for the reader's benefit.

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“…Thermostability is a key factor determining the practical application of enzymes at an industrial scale, since higher thermostability means a high durability of enzyme, and also the enzymatic reaction at higher temperature can accelerate the reaction rates (Yu and Huang 2014). Therefore, enhancing the thermostability has become one of the major fields in enzyme engineering (Ohta et al 2014).…”

Section: Introductionmentioning

confidence: 99%

Engineering the thermostability of β-glucuronidase from Penicillium purpurogenum Li-3 by loop transplant

Feng

Tang

Han

et al. 2016

Appl Microbiol Biotechnol

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In this study, we proposed a loop transplant strategy to improve the thermostability of Penicillium purpurogenum Li-3 β-glucuronidase expressed in Escherichia coli (abbreviated to PGUS-E). Firstly, three unstable surface loops of PGUS-E to be replaced were identified with regards to B-factor values and in-depth structure analysis: loops 205-211, 258-263, and 25-31. Then, based on B-factor analysis, eight stable loops for substitution were selected from two typical thermophilic glycosidases which had low homology with PGUS-E (less than 25 %). By analyzing the common features of these stable loops, it was found that they shared a common residue skeleton DXXTX(X)R, based on this, three chimera loops were also manually designed: RSQTSND, RSSTQRD, and DDQTSR. All these loops were introduced to replace the unstable loops of PGUS-E by homology structure modeling, and only mutants with increased hydrogen bonds number and good compatibility with the local mutated region were further subjected to experimental verification. By using this strategy, 10 mutants were experimentally generated, among which three mutants, M1, M3, and M8, were obtained which showed 11.8, 3.3, and 9.4 times higher half-life at 70 °C than that of wild-type (8.5 min). Finally, the MD simulation indicated that the increased hydrogen bonds, decreased flexibility of N-terminal, and increased π-π stacking interaction were responsible for the improved thermostability.

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Engineering proteins for thermostability through rigidifying flexible sites

Cited by 206 publications

References 88 publications

The role of proline substitutions within flexible regions on thermostability of luciferase

The role of proline substitutions within flexible regions on thermostability of luciferase

Genetically modified proteins: functional improvement and chimeragenesis

Engineering the thermostability of β-glucuronidase from Penicillium purpurogenum Li-3 by loop transplant

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