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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

Differences in the stability of rigid contacts between wild type and mutants of BsLipA.Maps depict differences between stability maps of the respective mutants and an average stability map of the six wild type structures (see the main text for explanation) for A: mutant 1-14F5, B: mutant 6B, and C: mutant X. A red (blue) color indicates that a rigid contact in the mutant is more (less) stable than in the wild type (see color scale at the bottom). The upper triangles show differences in the stability values for all residue pairs; the lower triangles show differences in the stability values only for residue pairs that are within 5 Å of each other, with values for all other residue pairs colored gray. Secondary structure elements as computed by the DSSP program [88, 89] are indicated on both abscissa and ordinate and are labeled: α-helix (red rectangle), β-strands (green rectangle), loop (black line). Arrows represent the mutation positions with respect to the wild type sequence: Common mutations in 1-14F5 (A) and 6B (B) are shown in magenta, unique mutations in 6B (B) are shown in green, and mutations in X (C) are shown in orange. The differences in the stability of rigid contacts for residue neighbors is also displayed on the structures of the mutants by sticks connecting Cα atoms of residue pairs colored according to the color scale of the maps for D: 1-14F5, E: 6B, and F: X. Only those contacts that are stabilized by ≥ 4 K or destabilized by ≥ 3 K are shown for clarity; for the same reason, contacts between two residues of the same secondary structure element are not shown. Mutated residues are shown as sticks and a sphere at their Cα atoms (D, E, and F) in the same color used for arrows (A, B, and C).
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pone.0130289.g006: Differences in the stability of rigid contacts between wild type and mutants of BsLipA.Maps depict differences between stability maps of the respective mutants and an average stability map of the six wild type structures (see the main text for explanation) for A: mutant 1-14F5, B: mutant 6B, and C: mutant X. A red (blue) color indicates that a rigid contact in the mutant is more (less) stable than in the wild type (see color scale at the bottom). The upper triangles show differences in the stability values for all residue pairs; the lower triangles show differences in the stability values only for residue pairs that are within 5 Å of each other, with values for all other residue pairs colored gray. Secondary structure elements as computed by the DSSP program [88, 89] are indicated on both abscissa and ordinate and are labeled: α-helix (red rectangle), β-strands (green rectangle), loop (black line). Arrows represent the mutation positions with respect to the wild type sequence: Common mutations in 1-14F5 (A) and 6B (B) are shown in magenta, unique mutations in 6B (B) are shown in green, and mutations in X (C) are shown in orange. The differences in the stability of rigid contacts for residue neighbors is also displayed on the structures of the mutants by sticks connecting Cα atoms of residue pairs colored according to the color scale of the maps for D: 1-14F5, E: 6B, and F: X. Only those contacts that are stabilized by ≥ 4 K or destabilized by ≥ 3 K are shown for clarity; for the same reason, contacts between two residues of the same secondary structure element are not shown. Mutated residues are shown as sticks and a sphere at their Cα atoms (D, E, and F) in the same color used for arrows (A, B, and C).

Mentions: Considering that the average local stability defined above correctly reflects differences in the (macroscopic) thermodynamic thermostability, we analyzed on a residue basis how changes in thermostability relate to changes in local structural stability (rigidity). First, we compared stability maps of variants from Rao et al. with distinct thermostabilities to analyze the effect of mutations on the local rigidity. In particular, we compared the wild type to a more thermostable variant 1-14F5 and the most thermostable variant 6B. We averaged stability maps of the six wild type structures (see above and Fig 4B) and used this average for comparison against the thermostable variants of BsLipA. Difference stability maps for 1-14F5/wild type (Fig 6A) and 6B/wild type (Fig 6B) pairs demonstrate that mutations in general improve the strength of rigid contacts to and in between neighboring residues of the mutations (lower triangles in Fig 6A and 6B) but also in between residue pairs not in contact distance (upper triangles in Fig 6A and 6B). This effect is more pronounced for 6B/wild type than 1-14F5/wild type.


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

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

Differences in the stability of rigid contacts between wild type and mutants of BsLipA.Maps depict differences between stability maps of the respective mutants and an average stability map of the six wild type structures (see the main text for explanation) for A: mutant 1-14F5, B: mutant 6B, and C: mutant X. A red (blue) color indicates that a rigid contact in the mutant is more (less) stable than in the wild type (see color scale at the bottom). The upper triangles show differences in the stability values for all residue pairs; the lower triangles show differences in the stability values only for residue pairs that are within 5 Å of each other, with values for all other residue pairs colored gray. Secondary structure elements as computed by the DSSP program [88, 89] are indicated on both abscissa and ordinate and are labeled: α-helix (red rectangle), β-strands (green rectangle), loop (black line). Arrows represent the mutation positions with respect to the wild type sequence: Common mutations in 1-14F5 (A) and 6B (B) are shown in magenta, unique mutations in 6B (B) are shown in green, and mutations in X (C) are shown in orange. The differences in the stability of rigid contacts for residue neighbors is also displayed on the structures of the mutants by sticks connecting Cα atoms of residue pairs colored according to the color scale of the maps for D: 1-14F5, E: 6B, and F: X. Only those contacts that are stabilized by ≥ 4 K or destabilized by ≥ 3 K are shown for clarity; for the same reason, contacts between two residues of the same secondary structure element are not shown. Mutated residues are shown as sticks and a sphere at their Cα atoms (D, E, and F) in the same color used for arrows (A, B, and C).
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Related In: Results  -  Collection

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

pone.0130289.g006: Differences in the stability of rigid contacts between wild type and mutants of BsLipA.Maps depict differences between stability maps of the respective mutants and an average stability map of the six wild type structures (see the main text for explanation) for A: mutant 1-14F5, B: mutant 6B, and C: mutant X. A red (blue) color indicates that a rigid contact in the mutant is more (less) stable than in the wild type (see color scale at the bottom). The upper triangles show differences in the stability values for all residue pairs; the lower triangles show differences in the stability values only for residue pairs that are within 5 Å of each other, with values for all other residue pairs colored gray. Secondary structure elements as computed by the DSSP program [88, 89] are indicated on both abscissa and ordinate and are labeled: α-helix (red rectangle), β-strands (green rectangle), loop (black line). Arrows represent the mutation positions with respect to the wild type sequence: Common mutations in 1-14F5 (A) and 6B (B) are shown in magenta, unique mutations in 6B (B) are shown in green, and mutations in X (C) are shown in orange. The differences in the stability of rigid contacts for residue neighbors is also displayed on the structures of the mutants by sticks connecting Cα atoms of residue pairs colored according to the color scale of the maps for D: 1-14F5, E: 6B, and F: X. Only those contacts that are stabilized by ≥ 4 K or destabilized by ≥ 3 K are shown for clarity; for the same reason, contacts between two residues of the same secondary structure element are not shown. Mutated residues are shown as sticks and a sphere at their Cα atoms (D, E, and F) in the same color used for arrows (A, B, and C).
Mentions: Considering that the average local stability defined above correctly reflects differences in the (macroscopic) thermodynamic thermostability, we analyzed on a residue basis how changes in thermostability relate to changes in local structural stability (rigidity). First, we compared stability maps of variants from Rao et al. with distinct thermostabilities to analyze the effect of mutations on the local rigidity. In particular, we compared the wild type to a more thermostable variant 1-14F5 and the most thermostable variant 6B. We averaged stability maps of the six wild type structures (see above and Fig 4B) and used this average for comparison against the thermostable variants of BsLipA. Difference stability maps for 1-14F5/wild type (Fig 6A) and 6B/wild type (Fig 6B) pairs demonstrate that mutations in general improve the strength of rigid contacts to and in between neighboring residues of the mutations (lower triangles in Fig 6A and 6B) but also in between residue pairs not in contact distance (upper triangles in Fig 6A and 6B). This effect is more pronounced for 6B/wild type than 1-14F5/wild type.

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