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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

Average loss of structural rigidity of the wild type BsLipA during a thermal unfolding simulation.Rigid clusters are depicted as uniformly colored bodies, with the largest rigid cluster shown in blue and smaller rigid clusters in the order of the colors green, magenta, cyan, orange, and violet. Temperatures are indicated for each depiction of a rigid cluster decomposition. At the beginning of the thermal unfolding simulation (302 K), almost the complete structure is part of the giant rigid cluster; in contrast, the structure becomes completely flexible at temperatures ≥374 K. The right views differ from the left ones by an anti-clockwise rotation of ~90° about a horizontal axis. Important secondary structure elements are labeled. Note that the unfolding pathway shown here represents an average loss of rigidity percolation calculated from a stability map (see section “Local and global rigidity indices” in S1 File) averaged over all unfolding trajectories obtained for the ensemble of 2000 network topologies. Hence, the temperature at the phase transition point identified that way (Figure B in S1 File) cannot be compared to the average phase transition temperature, which is obtained from 2000 individual Tp values and used for predicting the thermodynamic thermostability of BsLipA variants (see section “Prediction of thermodynamic thermostability of BsLipA variants”)
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pone.0130289.g003: Average loss of structural rigidity of the wild type BsLipA during a thermal unfolding simulation.Rigid clusters are depicted as uniformly colored bodies, with the largest rigid cluster shown in blue and smaller rigid clusters in the order of the colors green, magenta, cyan, orange, and violet. Temperatures are indicated for each depiction of a rigid cluster decomposition. At the beginning of the thermal unfolding simulation (302 K), almost the complete structure is part of the giant rigid cluster; in contrast, the structure becomes completely flexible at temperatures ≥374 K. The right views differ from the left ones by an anti-clockwise rotation of ~90° about a horizontal axis. Important secondary structure elements are labeled. Note that the unfolding pathway shown here represents an average loss of rigidity percolation calculated from a stability map (see section “Local and global rigidity indices” in S1 File) averaged over all unfolding trajectories obtained for the ensemble of 2000 network topologies. Hence, the temperature at the phase transition point identified that way (Figure B in S1 File) cannot be compared to the average phase transition temperature, which is obtained from 2000 individual Tp values and used for predicting the thermodynamic thermostability of BsLipA variants (see section “Prediction of thermodynamic thermostability of BsLipA variants”)

Mentions: From monitoring the loss in rigidity percolation during thermal unfolding simulations, major phase transitions in the protein can be identified that relate to the unfolding pathway [11–13, 54, 73]. Here, we describe the loss of rigidity percolation of the wild type BsLipA (PDB ID 1ISP) as an example. Similarity or dissimilarity, respectively, of the unfolding pathways across all variants is described below. During the thermal unfolding, a giant rigid cluster that exists at low temperature (equivalent to a high Ecut) breaks down in smaller sub-clusters until, finally, the whole protein becomes flexible at a high temperature (Fig 3; see also S1 Video showing the loss of rigidity percolation during the thermal unfolding of the wild type). As such, nearly the entire protein structure constitutes a single giant rigid cluster initially (at 302 K; Fig 3). As the temperature increases, loops segregate first from the giant rigid cluster. Then, at 314 K, α-helix D (αD) and αE segregate to form individual small rigid clusters (Fig 3), as do αA and αF at 318 K. The giant rigid cluster at this temperature is formed by the central β-sheet region and the two helices αB and αC (Fig 3). Next, the β-sheet region becomes sequentially flexible, beginning with β4 and β8 at 320 K (Fig 3). Then, the remaining β-strands become flexible in the order β3, β7, and β5−β6, leading to a completely flexible β-sheet region at 332 K (Fig 3 The immediate next step at which αB and αC become two separate rigid clusters is identified as a phase transition point: Now most of the structure has become flexible. This transition is most prominent with respect to going from a structurally stable wild type BsLipA to an unfolded one (Figure B in S1 File). After this phase transition point, the remaining rigidity is sequentially lost, and the structure finally becomes completely flexible at 374 K (Fig 3).


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

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

Average loss of structural rigidity of the wild type BsLipA during a thermal unfolding simulation.Rigid clusters are depicted as uniformly colored bodies, with the largest rigid cluster shown in blue and smaller rigid clusters in the order of the colors green, magenta, cyan, orange, and violet. Temperatures are indicated for each depiction of a rigid cluster decomposition. At the beginning of the thermal unfolding simulation (302 K), almost the complete structure is part of the giant rigid cluster; in contrast, the structure becomes completely flexible at temperatures ≥374 K. The right views differ from the left ones by an anti-clockwise rotation of ~90° about a horizontal axis. Important secondary structure elements are labeled. Note that the unfolding pathway shown here represents an average loss of rigidity percolation calculated from a stability map (see section “Local and global rigidity indices” in S1 File) averaged over all unfolding trajectories obtained for the ensemble of 2000 network topologies. Hence, the temperature at the phase transition point identified that way (Figure B in S1 File) cannot be compared to the average phase transition temperature, which is obtained from 2000 individual Tp values and used for predicting the thermodynamic thermostability of BsLipA variants (see section “Prediction of thermodynamic thermostability of BsLipA variants”)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493141&req=5

pone.0130289.g003: Average loss of structural rigidity of the wild type BsLipA during a thermal unfolding simulation.Rigid clusters are depicted as uniformly colored bodies, with the largest rigid cluster shown in blue and smaller rigid clusters in the order of the colors green, magenta, cyan, orange, and violet. Temperatures are indicated for each depiction of a rigid cluster decomposition. At the beginning of the thermal unfolding simulation (302 K), almost the complete structure is part of the giant rigid cluster; in contrast, the structure becomes completely flexible at temperatures ≥374 K. The right views differ from the left ones by an anti-clockwise rotation of ~90° about a horizontal axis. Important secondary structure elements are labeled. Note that the unfolding pathway shown here represents an average loss of rigidity percolation calculated from a stability map (see section “Local and global rigidity indices” in S1 File) averaged over all unfolding trajectories obtained for the ensemble of 2000 network topologies. Hence, the temperature at the phase transition point identified that way (Figure B in S1 File) cannot be compared to the average phase transition temperature, which is obtained from 2000 individual Tp values and used for predicting the thermodynamic thermostability of BsLipA variants (see section “Prediction of thermodynamic thermostability of BsLipA variants”)
Mentions: From monitoring the loss in rigidity percolation during thermal unfolding simulations, major phase transitions in the protein can be identified that relate to the unfolding pathway [11–13, 54, 73]. Here, we describe the loss of rigidity percolation of the wild type BsLipA (PDB ID 1ISP) as an example. Similarity or dissimilarity, respectively, of the unfolding pathways across all variants is described below. During the thermal unfolding, a giant rigid cluster that exists at low temperature (equivalent to a high Ecut) breaks down in smaller sub-clusters until, finally, the whole protein becomes flexible at a high temperature (Fig 3; see also S1 Video showing the loss of rigidity percolation during the thermal unfolding of the wild type). As such, nearly the entire protein structure constitutes a single giant rigid cluster initially (at 302 K; Fig 3). As the temperature increases, loops segregate first from the giant rigid cluster. Then, at 314 K, α-helix D (αD) and αE segregate to form individual small rigid clusters (Fig 3), as do αA and αF at 318 K. The giant rigid cluster at this temperature is formed by the central β-sheet region and the two helices αB and αC (Fig 3). Next, the β-sheet region becomes sequentially flexible, beginning with β4 and β8 at 320 K (Fig 3). Then, the remaining β-strands become flexible in the order β3, β7, and β5−β6, leading to a completely flexible β-sheet region at 332 K (Fig 3 The immediate next step at which αB and αC become two separate rigid clusters is identified as a phase transition point: Now most of the structure has become flexible. This transition is most prominent with respect to going from a structurally stable wild type BsLipA to an unfolded one (Figure B in S1 File). After this phase transition point, the remaining rigidity is sequentially lost, and the structure finally becomes completely flexible at 374 K (Fig 3).

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