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Structural Rigidity and Protein Thermostability in Variants of Lipase A from Bacillus subtilis.

Rathi PC, Jaeger KE, Gohlke H - PLoS ONE (2015)

Bottom Line: Furthermore, we introduce a robust, local stability measure for predicting thermodynamic thermostability.Our results complement work that showed for pairs of homologous proteins that raising the structural stability is the most common way to obtain a higher thermostability.Furthermore, they demonstrate that related series of mutants with only a small number of mutations can be successfully analyzed by CNA, which suggests that CNA can be applied prospectively in rational protein design aimed at higher thermodynamic thermostability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical and Medical Chemistry, Heinrich-Heine-University, Düsseldorf, Germany.

ABSTRACT
Understanding the origin of thermostability is of fundamental importance in protein biochemistry. Opposing views on increased or decreased structural rigidity of the folded state have been put forward in this context. They have been related to differences in the temporal resolution of experiments and computations that probe atomic mobility. Here, we find a significant (p = 0.004) and fair (R2 = 0.46) correlation between the structural rigidity of a well-characterized set of 16 mutants of lipase A from Bacillus subtilis (BsLipA) and their thermodynamic thermostability. We apply the rigidity theory-based Constraint Network Analysis (CNA) approach, analyzing directly and in a time-independent manner the statics of the BsLipA mutants. We carefully validate the CNA results on macroscopic and microscopic experimental observables and probe for their sensitivity with respect to input structures. Furthermore, we introduce a robust, local stability measure for predicting thermodynamic thermostability. Our results complement work that showed for pairs of homologous proteins that raising the structural stability is the most common way to obtain a higher thermostability. Furthermore, they demonstrate that related series of mutants with only a small number of mutations can be successfully analyzed by CNA, which suggests that CNA can be applied prospectively in rational protein design aimed at higher thermodynamic thermostability.

No MeSH data available.


Related in: MedlinePlus

Cartoon representation of wild type BsLipA with mutated residues indicated by spheres of their Cα atoms (mutations from Rao et al. [44–48]: magenta; Reetz et al. [42, 49]: orange; mutations common in both data sets: cyan).The catalytic triad (Ser77-Asp133-His156) is shown in stick representation with yellow carbons. The protein is colored according to secondary structure (α-helices: red; β-sheets: yellow; loops: green). The right view (B) differs from the left (A) by an anti-clockwise rotation of ~90° about a horizontal axis. All figures of BsLipA structures were generated with PyMOL (http://www.pymol.org).
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pone.0130289.g002: Cartoon representation of wild type BsLipA with mutated residues indicated by spheres of their Cα atoms (mutations from Rao et al. [44–48]: magenta; Reetz et al. [42, 49]: orange; mutations common in both data sets: cyan).The catalytic triad (Ser77-Asp133-His156) is shown in stick representation with yellow carbons. The protein is colored according to secondary structure (α-helices: red; β-sheets: yellow; loops: green). The right view (B) differs from the left (A) by an anti-clockwise rotation of ~90° about a horizontal axis. All figures of BsLipA structures were generated with PyMOL (http://www.pymol.org).

Mentions: BsLipA is a protein of 181 amino acids with a minimal α/β hydrolase fold; in this fold, a central parallel β-sheet of six β-strands is surrounded by six α-helices. Ser77, Asp133, and His156 constitute the catalytic triad (Fig 2). Unlike other lipases, the catalytic site in BsLipA is not covered with a lid. Hence, BsLipA does not show interfacial activation [40]. The data set used in this study contains structures of the wild type BsLipA, thirteen mutants from Rao et al. [44–48], and three mutants from Reetz et al. [42, 49] (Table 1). The mutants differ from the wild type by three to twelve mutations, i.e., the sequence identity is > 93%. Models for the mutants for which X-ray structures were not available were built using the SCWRL program. As the number of mutations in the modeled variants is ≤ 7 with respect to the template structures (< 4% with respect to the sequence length) (Table 1), an overall similar backbone confirmation can be expected as can be an overall reliable modeling of side chain conformations by SCWRL. This was also evident from a very good structural alignment and low root-mean-square deviations (RMSD) between the wild type and those mutants for which crystal structures were available (Cα atom-based RMSD values between the wild type and the mutants < 0.38 Å). The high structural similarity allows a direct comparison of results from rigidity analyses for these structures [11–13].


Structural Rigidity and Protein Thermostability in Variants of Lipase A from Bacillus subtilis.

Rathi PC, Jaeger KE, Gohlke H - PLoS ONE (2015)

Cartoon representation of wild type BsLipA with mutated residues indicated by spheres of their Cα atoms (mutations from Rao et al. [44–48]: magenta; Reetz et al. [42, 49]: orange; mutations common in both data sets: cyan).The catalytic triad (Ser77-Asp133-His156) is shown in stick representation with yellow carbons. The protein is colored according to secondary structure (α-helices: red; β-sheets: yellow; loops: green). The right view (B) differs from the left (A) by an anti-clockwise rotation of ~90° about a horizontal axis. All figures of BsLipA structures were generated with PyMOL (http://www.pymol.org).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493141&req=5

pone.0130289.g002: Cartoon representation of wild type BsLipA with mutated residues indicated by spheres of their Cα atoms (mutations from Rao et al. [44–48]: magenta; Reetz et al. [42, 49]: orange; mutations common in both data sets: cyan).The catalytic triad (Ser77-Asp133-His156) is shown in stick representation with yellow carbons. The protein is colored according to secondary structure (α-helices: red; β-sheets: yellow; loops: green). The right view (B) differs from the left (A) by an anti-clockwise rotation of ~90° about a horizontal axis. All figures of BsLipA structures were generated with PyMOL (http://www.pymol.org).
Mentions: BsLipA is a protein of 181 amino acids with a minimal α/β hydrolase fold; in this fold, a central parallel β-sheet of six β-strands is surrounded by six α-helices. Ser77, Asp133, and His156 constitute the catalytic triad (Fig 2). Unlike other lipases, the catalytic site in BsLipA is not covered with a lid. Hence, BsLipA does not show interfacial activation [40]. The data set used in this study contains structures of the wild type BsLipA, thirteen mutants from Rao et al. [44–48], and three mutants from Reetz et al. [42, 49] (Table 1). The mutants differ from the wild type by three to twelve mutations, i.e., the sequence identity is > 93%. Models for the mutants for which X-ray structures were not available were built using the SCWRL program. As the number of mutations in the modeled variants is ≤ 7 with respect to the template structures (< 4% with respect to the sequence length) (Table 1), an overall similar backbone confirmation can be expected as can be an overall reliable modeling of side chain conformations by SCWRL. This was also evident from a very good structural alignment and low root-mean-square deviations (RMSD) between the wild type and those mutants for which crystal structures were available (Cα atom-based RMSD values between the wild type and the mutants < 0.38 Å). The high structural similarity allows a direct comparison of results from rigidity analyses for these structures [11–13].

Bottom Line: Furthermore, we introduce a robust, local stability measure for predicting thermodynamic thermostability.Our results complement work that showed for pairs of homologous proteins that raising the structural stability is the most common way to obtain a higher thermostability.Furthermore, they demonstrate that related series of mutants with only a small number of mutations can be successfully analyzed by CNA, which suggests that CNA can be applied prospectively in rational protein design aimed at higher thermodynamic thermostability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical and Medical Chemistry, Heinrich-Heine-University, Düsseldorf, Germany.

ABSTRACT
Understanding the origin of thermostability is of fundamental importance in protein biochemistry. Opposing views on increased or decreased structural rigidity of the folded state have been put forward in this context. They have been related to differences in the temporal resolution of experiments and computations that probe atomic mobility. Here, we find a significant (p = 0.004) and fair (R2 = 0.46) correlation between the structural rigidity of a well-characterized set of 16 mutants of lipase A from Bacillus subtilis (BsLipA) and their thermodynamic thermostability. We apply the rigidity theory-based Constraint Network Analysis (CNA) approach, analyzing directly and in a time-independent manner the statics of the BsLipA mutants. We carefully validate the CNA results on macroscopic and microscopic experimental observables and probe for their sensitivity with respect to input structures. Furthermore, we introduce a robust, local stability measure for predicting thermodynamic thermostability. Our results complement work that showed for pairs of homologous proteins that raising the structural stability is the most common way to obtain a higher thermostability. Furthermore, they demonstrate that related series of mutants with only a small number of mutations can be successfully analyzed by CNA, which suggests that CNA can be applied prospectively in rational protein design aimed at higher thermodynamic thermostability.

No MeSH data available.


Related in: MedlinePlus