The effects of amino acid replacements of glycine 20 on conformational stability and catalysis of staphylococcal nuclease

Feng, Yajuan; Huang, Sun; Zhang, Wenzheng; Zeng, Z.; Zou, Xiajuan; Zhong, Liang; Peng, Jiarou; Jing, Guozhong

doi:10.1016/j.biochi.2004.10.005

Cited by 6 publications

(7 citation statements)

References 35 publications

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“…Glycine, the only amino acid that lacks a β‐carbon, has the highest conformational entropy [18], while proline can adopt only a few configurations and has the lowest conformational entropy [19,20]. A glycine to proline mutation could therefore decrease the conformational entropy of a protein and lead to stabilization [21–25].…”

Section: Introductionmentioning

confidence: 99%

Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation

et al. 2010

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Protein thermostability can be increased by some glycine to proline mutations in a target protein. However, not all glycine to proline mutations can improve protein thermostability, and this method is suitable only at carefully selected mutation sites that can accommodate structural stabilization. In this study, homology modeling and molecular dynamics simulations were used to select appropriate glycine to proline mutations to improve protein thermostability, and the effect of the selected mutations was proved by the experiments. The structure of methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 (Ochr‐MPH) was constructed by homology modeling, and molecular dynamics simulations were performed on the modeled structure. A profile of the root mean square fluctuations of Ochr‐MPH was calculated at the nanosecond timescale, and an eight‐amino acid loop region (residues 186–193) was identified as having high conformational fluctuation. The two glycines nearest to this region were selected as mutation targets that might affect protein flexibility in the vicinity. The structures and conformational fluctuations of two single mutants (G194P and G198P) and one double mutant (G194P/G198P) were modeled and analyzed using molecular dynamics simulations. The results predicted that the mutant G194P had the decreased conformational fluctuation in the loop region and might increase the thermostability of Ochr‐MPH. The thermostability and kinetic behavior of the wild‐type and three mutant enzymes were measured. The results were consistent with the computational predictions, and the mutant G194P was found to have higher thermostability than the wild‐type enzyme.

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Section: Introductionmentioning

confidence: 99%

Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation

et al. 2010

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“…The entropic stabilization is likely responsible for the stabilization of Zone 3 caused by the G67A mutation. Glycine is the only amino acid that lacks a β-carbon and the rotation of N–Cα is rarely restricted, contributing to a large degree of conformational freedom and a decrease of conformational rigidity. , Compared with glycine, alanine has higher space steric hindrance and lower conformational entropy, which limits the possible backbone conformation and stabilizes the folded protein. − …”

Section: Resultsmentioning

confidence: 99%

“…Glycine is the only amino acid that lacks a β-carbon and the rotation of N−Cα is rarely restricted, contributing to a large degree of conformational freedom and a decrease of conformational rigidity. 40,51 Compared with glycine, alanine has higher space steric hindrance and lower conformational entropy, which limits the possible backbone conformation and stabilizes the folded protein. 52−54 In contrast to other zones, there were three mutations in Zone 4 (N290E, E294S, S303A), implying that the residue interactions in this region may be very variable.…”

Section: Simulation and Structural Analysis Of Stabilized Mutantsmentioning

confidence: 99%

Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

et al. 2023

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The flexible regions represented by loops have been the focus of protein stability engineering. However, residues located in specific rigid regions are also crucial for protein stability. The role of dynamic correlations between mutations in these two regions on protein stabilization remains inadequately understood. In this work, a ketoreductase CpKR originated from Candida parapsilosis was rationally engineered to improve its stability by comprehensively redesigning the flexible and rigid regions. Hot spots were successively identified and validated in the rigid regions with low B-factor or root-mean-square fluctuation values, as well as in relatively flexible areas of the protein. Based on the non-additive and cooperative mutational effects, a combinatorial variant (M2) with beneficial mutations in these two distinct regions showed a 4630-fold increased half-life at 40 °C and a 12 °C higher apparent melting temperature as compared with those of the wild type. The variant M2 also showed improved pH compatibility, better organic substance tolerance, and enhanced performance in asymmetric reduction of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxo-propanoate (1a), the precursor of cardiovascular drug Diltiazem. The crystal structures and molecular dynamics simulations demonstrated that additional interaction networks in both the rigid and flexible regions modulated protein stability, and the dynamic correlation between these two regions mediated the observed synergistic combination. These results showed that both the flexible and rigid regions, especially the cross-correlated areas, are crucial for enhancing protein stability, and comprehensively redesigning these two regions is a powerful and attractive approach for acquiring stable enzymes.

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“…SNase is a protein that is widely studied for protein folding, [28][29][30][31] stability, [32,33] dynamics, [34,35] and catalysis activity. [36] SNase, which comes from staphylococcus aureus, is a small globular protein with a single chain and a single domain. Its molecular weight is 16800 kDa, with 149 amino acids, including one Trp residue at site 140.…”

Section: Snase Tryptophan and Mutagenesismentioning

confidence: 99%

“…[36] SNase, which comes from staphylococcus aureus, is a small globular protein with single chain and single domain. Its molecular weight is 16800kDa, with 149 amino acid including one Trp residue at site 140.…”

Section: Snase Tryptophan and Mutagenesismentioning

confidence: 99%

Ultrafast solvation dynamics at internal sites of staphylococcal nuclease investigated by site-directed mutagenesis

Gao¹,

Li²,

Wang³

et al. 2015

Chinese Phys. B

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Solvation is essential for protein activities. To study internal solvation of protein, site-directed mutagenesis is applied. Intrinsic fluorescent probe, tryptophan, is inserted into desired position inside protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics researches. We introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside caves of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at internal sites of the caves indicate clear characteristics of local environment. These solvation behaviors correlated to the enzyme activity directly.

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The effects of amino acid replacements of glycine 20 on conformational stability and catalysis of staphylococcal nuclease

Cited by 6 publications

References 35 publications

Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation

Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation

Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

Ultrafast solvation dynamics at internal sites of staphylococcal nuclease investigated by site-directed mutagenesis

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