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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

Correlation between predicted and experimental thermostabilities (Tm values) of BsLipA variants; for the predictions, the ENTFNC approach was used.A: Correlation between Tp derived from the global index Htype2 and Tm values for thermodynamically thermostabilized mutants from Rao et al. Data points colored red were considered outliers (see main text for explanation) and excluded when calculating R2 values and the correlation lines. B: Correlation between  and Tm values for thermodynamically thermostabilized mutants from Rao et al. Data points shown as empty squares represent  values for five additional wild type crystal structures (see main text for details; two of the squares closely overlap; mean  over all six data points for wild type structures is shown as a small horizontal line: 315.9 ± 0.6 K). A and B: Error bars represent the standard error in the mean. Tp and  values for kinetically thermostabilized mutants from Reetz et al. are marked by arrows on the corresponding ordinates.
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pone.0130289.g004: Correlation between predicted and experimental thermostabilities (Tm values) of BsLipA variants; for the predictions, the ENTFNC approach was used.A: Correlation between Tp derived from the global index Htype2 and Tm values for thermodynamically thermostabilized mutants from Rao et al. Data points colored red were considered outliers (see main text for explanation) and excluded when calculating R2 values and the correlation lines. B: Correlation between and Tm values for thermodynamically thermostabilized mutants from Rao et al. Data points shown as empty squares represent values for five additional wild type crystal structures (see main text for details; two of the squares closely overlap; mean over all six data points for wild type structures is shown as a small horizontal line: 315.9 ± 0.6 K). A and B: Error bars represent the standard error in the mean. Tp and values for kinetically thermostabilized mutants from Reetz et al. are marked by arrows on the corresponding ordinates.

Mentions: From the thermal unfolding simulations, the temperature of the phase transition point Tp was identified as described in the section “Local and global rigidity indices” in S1 File. Note that Tp values determined that way should be considered relative values only, as stated in previous studies [12, 34, 35]. Initially, we calculated phase transition points using single network topologies generated from the input structures of wild type BsLipA and mutants of Rao et al.; however, this resulted in a very poor prediction of thermodynamic thermostability with a coefficient of determination (R2) for a linear fit between experimental Tm and predicted Tp of 0.22 (Figure C in S1 File). We anticipated that this result reflects the high sensitivity of CNA on the conformation of the input structures as also found previously [11, 56, 64, 65]. We thus resorted to averaging Tp values over an ensemble of BsLipA, applying the recently developed ENTFNC approach. This approach generates an ensemble of network topologies from a single input structure and has been shown to yield results of rigidity analyses both at the local and global level that agree almost perfectly with those obtained from MD simulations-generated ensembles of structures [56]. However, this yielded a significant (p = 0.002) correlation between Tp and Tm with R2 = 0.58 only if the two structures with the lowest (wild type) and highest (mutant 6B) Tm were considered outliers (Fig 4A; see below for an explanation regarding the outliers; note that removing the two outliers in the case of using single network topologies only marginally improved R2 from 0.22 to 0.29). The mutants IX, X and XI of Reetz et al. were predicted to be slightly less thermostable than the wild type (Fig 4A). This is in line with experimental findings by Reetz et al. that suggest that these mutants are thermodynamically less stable than the wild type [49]. In summary, these results suggest that CNA coupled with the ENTFNC approach can sense effects on the thermodynamic thermostability that arise from only a few sequence variations (pairwise sequence identity > 93%; pairwise RMSD < 0.38 Å). However, the false predictions for wild type BsLipA and mutant 6B are dissatisfying.


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

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

Correlation between predicted and experimental thermostabilities (Tm values) of BsLipA variants; for the predictions, the ENTFNC approach was used.A: Correlation between Tp derived from the global index Htype2 and Tm values for thermodynamically thermostabilized mutants from Rao et al. Data points colored red were considered outliers (see main text for explanation) and excluded when calculating R2 values and the correlation lines. B: Correlation between  and Tm values for thermodynamically thermostabilized mutants from Rao et al. Data points shown as empty squares represent  values for five additional wild type crystal structures (see main text for details; two of the squares closely overlap; mean  over all six data points for wild type structures is shown as a small horizontal line: 315.9 ± 0.6 K). A and B: Error bars represent the standard error in the mean. Tp and  values for kinetically thermostabilized mutants from Reetz et al. are marked by arrows on the corresponding ordinates.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130289.g004: Correlation between predicted and experimental thermostabilities (Tm values) of BsLipA variants; for the predictions, the ENTFNC approach was used.A: Correlation between Tp derived from the global index Htype2 and Tm values for thermodynamically thermostabilized mutants from Rao et al. Data points colored red were considered outliers (see main text for explanation) and excluded when calculating R2 values and the correlation lines. B: Correlation between and Tm values for thermodynamically thermostabilized mutants from Rao et al. Data points shown as empty squares represent values for five additional wild type crystal structures (see main text for details; two of the squares closely overlap; mean over all six data points for wild type structures is shown as a small horizontal line: 315.9 ± 0.6 K). A and B: Error bars represent the standard error in the mean. Tp and values for kinetically thermostabilized mutants from Reetz et al. are marked by arrows on the corresponding ordinates.
Mentions: From the thermal unfolding simulations, the temperature of the phase transition point Tp was identified as described in the section “Local and global rigidity indices” in S1 File. Note that Tp values determined that way should be considered relative values only, as stated in previous studies [12, 34, 35]. Initially, we calculated phase transition points using single network topologies generated from the input structures of wild type BsLipA and mutants of Rao et al.; however, this resulted in a very poor prediction of thermodynamic thermostability with a coefficient of determination (R2) for a linear fit between experimental Tm and predicted Tp of 0.22 (Figure C in S1 File). We anticipated that this result reflects the high sensitivity of CNA on the conformation of the input structures as also found previously [11, 56, 64, 65]. We thus resorted to averaging Tp values over an ensemble of BsLipA, applying the recently developed ENTFNC approach. This approach generates an ensemble of network topologies from a single input structure and has been shown to yield results of rigidity analyses both at the local and global level that agree almost perfectly with those obtained from MD simulations-generated ensembles of structures [56]. However, this yielded a significant (p = 0.002) correlation between Tp and Tm with R2 = 0.58 only if the two structures with the lowest (wild type) and highest (mutant 6B) Tm were considered outliers (Fig 4A; see below for an explanation regarding the outliers; note that removing the two outliers in the case of using single network topologies only marginally improved R2 from 0.22 to 0.29). The mutants IX, X and XI of Reetz et al. were predicted to be slightly less thermostable than the wild type (Fig 4A). This is in line with experimental findings by Reetz et al. that suggest that these mutants are thermodynamically less stable than the wild type [49]. In summary, these results suggest that CNA coupled with the ENTFNC approach can sense effects on the thermodynamic thermostability that arise from only a few sequence variations (pairwise sequence identity > 93%; pairwise RMSD < 0.38 Å). However, the false predictions for wild type BsLipA and mutant 6B are dissatisfying.

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