- Open Access
Predicting changes in protein thermostability brought about by single- or multi-site mutations
© Tian et al; licensee BioMed Central Ltd. 2010
- Received: 29 March 2010
- Accepted: 2 July 2010
- Published: 2 July 2010
An important aspect of protein design is the ability to predict changes in protein thermostability arising from single- or multi-site mutations. Protein thermostability is reflected in the change in free energy (ΔΔG) of thermal denaturation.
We have developed predictive software, Prethermut, based on machine learning methods, to predict the effect of single- or multi-site mutations on protein thermostability. The input vector of Prethermut is based on known structural changes and empirical measurements of changes in potential energy due to protein mutations. Using a 10-fold cross validation test on the M-dataset, consisting of 3366 mutants proteins from ProTherm, the classification accuracy of random forests and the regression accuracy of random forest regression were slightly better than support vector machines and support vector regression, whereas the overall accuracy of classification and the Pearson correlation coefficient of regression were 79.2% and 0.72, respectively. Prethermut performs better on proteins containing multi-site mutations than those with single mutations.
The performance of Prethermut indicates that it is a useful tool for predicting changes in protein thermostability brought about by single- or multi-site mutations and will be valuable in the rational design of proteins.
- Support Vector Machine
- Random Forest
- Support Vector Regression
- Reliability Index
- Machine Learning Method
Improving protein thermostability is an important goal of protein engineering ; by making enzymes easier to handle, increased thermostability can increase storage options and expand the temperature range of applications, and facilitate the commercial development of enzymatic products [2–5]. Mutations at certain residues can significantly alter a protein's structure and thermostability [6, 7]. The free energy (ΔG) of denaturation can be altered by single- or multi-site mutations; the change in ΔG (ΔΔG), an indication of the change in protein thermostability, has been determined for many mutated proteins by the thermal denaturation method . These data have been collected and deposited in publicly available databases [6, 8, 9]. From these data it is possible to develop computational methods to identify mutations in silico that could improve protein thermostability.
Various methods [2, 10] have been proposed to predict thermostability changes brought about by protein mutations; these methods have been based on changes in structural energy [4, 11, 12], statistical analyses of mutant protein thermostability [13, 14], and machine learning [15–21]. Methods based on changes in structural energy typically attempt to analyze changes in physical energy potentials , either by calculation, statistical analysis, or empirical measurement, with the objective of understanding the effects of mutations by comparing the energy difference between the wild-type and mutant structures [22, 23].
Recently, various machine learning approaches based on support vector machines (SVM) [18, 19, 24], neural networks , and decision trees  have been proposed for predicting the effects of mutations on thermostability [8, 9]. These approaches typically use large datasets of known primary, secondary, and tertiary structures of proteins to train the complex nonlinear functions.
Most approaches to predicting stability changes caused by mutations focus on a small number of mutations in a protein, often at a single site [2, 23]. However, many factors, such as hydrophobicity, van der Waals interactions, hydrogen bonds, ion pairs, and non-covalent interactions, contribute to protein thermostability . Thus, multi-site mutations would typically be expected to have a greater and more complex effect on protein thermostability than can be determined from single-site mutations alone [26, 27].
It is thus necessary to have a reliable method for discriminating between stabilizing and destabilizing mutations, as well as for predicting the effects of single- and multi-site mutations on the thermostability of proteins. In this study, we introduce the program "Prethermut" (Pre dicting changes in protein ther mostability brought about by single- or multi-site mu tations), which predicts protein thermostability changes caused by single- or multi-site mutations. The program uses machine learning to construct classification models (for predicting only the sign of ΔΔG) and regression models (for predicting the actual value of ΔΔG). The input feature of Prethermut was developed from structural energy calculations derived from empirical measurements of energy potentials and certain structural attributes reflecting non-covalent interactions between residues within the 3-D structure. Two large non-redundant datasets, the M-dataset and S-dataset, were used to train Prethermut and test its robustness, respectively.
Training and validation
Classification and regression performance of Prethermut on the M-dataset
Structural features used in Prethermut
Stereochemical improper dihedral potential
Van der Waals forces
Van der Waals clashes
Entropy side chain
Entropy main chain
The classifiers of random forests (RF) and support vector machines (SVM) were trained on the M-dataset to predict whether the mutations were stabilizing or destabilizing (i.e., the sign of ΔΔG). The regression methods of random forest regression (RFR) and support vector regression (SVR) were used to predict the change in free energy (ΔΔG) of thermal denaturation of the mutant proteins. Because the number of mutants in the training set having increased thermostability was disproportionately small versus those with decreased thermostability (by a factor of approximately three), the down sampling approach  was used for RF implementation, and for SVM implementation the weight given to the mutants with increased thermostability was 3-fold greater than that given to the mutants with decreased thermostability. The performance of the methods was assessed by a 10-fold cross validation on the M-dataset (Table 1).
The importance of each variable to the input vector of Prethermut was also assessed by evaluating the decrease in the classification accuracy of RF [30, 31]. As shown in Additional file 1: Table S1, all structural features contributed to the predictor, with the most important feature being the total energy, as calculated by FoldX [11, 28].
Prediction accuracy using different numbers of mutation sites
Performance of Prethermut on the M-dataset with different ranges of absolute ΔΔG
Range of absolute ΔΔG
Reliability index of classification by Prethermut
Comparison with other methods
Performance of Prethermut and other computational methods on the S-dataset
Several predictors [2, 16–18] have been constructed to predict the effect of a single mutation on protein thermostability, based on structural or sequence features. However, multi-site mutations usually have a greater effect on protein thermostability than single-site mutations . In this study, we present a predictive computer program, called Prethermut, based on machine learning methods, that can directly predict the effect of single- and multi-site mutations on protein thermostability from the wild-type protein's structural features.
The high predictive power of Prethermut, assessed by a rigorous 10-fold cross validation procedure, is illustrated by a Q2 value of 79.7% for the classification of stabilizing and destabilizing mutations from the M-dataset, and a Pearson's r of 0.79 for the correlation between predicted and experimentally determined ΔΔG values. The performance of Prethermut was also assessed in the independent S-dataset of more than 2000 mutants. Prethermut outperformed several published structure- and sequence-based predictors using the S-dataset. Although direct comparison of Prethermut with the other published predictors is not appropriate, because of differences in datasets used for training and testing, as well as the information used to develop the models, the results indicate that Prethermut is a powerful tool for predicting the effect of mutations on protein thermostability.
Both single- and multi-site mutations were considered.
The protein structure was known at atomic resolution and had been deposited in the Protein Data Bank.
Redundant data were removed, and an average free energy change (ΔΔG) of the mutant was calculated when multiple data for the mutant, using the same experimental procedure, were available.
The final non-redundant M-dataset consisted of 3366 mutants with single- or multi-site mutations acquired from 129 different proteins. The ΔΔG ranged from -12.23 kcal/mol to 13.7 kcal/mol. This dataset is available at http://www.mobioinfor.cn/prethermut/download.htm.
The second dataset, the S-dataset, was compiled by Dr. Vladimir Potapov  and obtained from http://ligin.weizmann.ac.il/~lpotapov/PEDS_mutants/mutants.html. This large, non-redundant dataset contained 2156 single-site mutants from 84 different proteins.
Input vectors and encoding schemes
For each mutant represented in the dataset, a wild-type structure was downloaded from the Protein Data Bank. All water molecules in the structures were manually removed.
Structures of the mutants were modelled by the program Modeller 9.7, which uses the standard steps for building mutants . It was supposed that k different structures of a mutant were modelled by Modeller 9.7 , as the Modeller program generates different mutant structures based on different random seeds .
For training the model, the input vector of Prethermut contained 58 elements that were calculated from 29 structural features (Table 2), including the potential energies and physical characteristics of the features. In this study, the programs FoldX 3.0  and Modeller 9.7 were used to calculate the values for these features, because these two programs have been widely used to predict and assess protein structures and are freely available.
For each mutant, k different structures were modeled. The input vector M i of the i th structure of the mutant was then calculated by FoldX 3.0 and Modeller 9.7, M i = [ ], where the value of i is from 1 to k. Then, all of the vectors M were averaged to yield a new vector N, N= [g 1 , g 2 ...g 29 ].
All of the residues in the wild-type protein were mutated via single site saturation mutagenesis by Modeller 9.7 . It was supposed that the length of the wild-type protein is l, and then l × 19 mutants for the wild-type protein would be modeled by Modeller 9.7. The structural feature vector W j = [ ] for the j th structure in all of the mutants of the wild-type protein was calculated, where the value of j is from 1 to l*19. The mean vector U= [μ 1 , μ 2 ...μ 29 ] and standard deviation vector S= [σ 1 , σ 2 ...σ 29 ] for all of the structural feature vectors W j (j = 1, 2...l*20) were calculated.
Machine learning methods
RF. The RF is an ensemble machine learning methodology originated by Leo Breiman . The basic idea of ensemble learning is to boost the performance of a number of weak learners via a voting scheme, where a weak learner can be an individual decision tree, a single perceptron/sigmoid function, or other simple and fast classifier . Regarding RF, its hallmarks include bootstrap re-sampling, random feature selection, in-depth decision tree construction and out-of-bag error estimates .
SVM. SVM is used to identify the optimal hyperplane that separates two classes of samples [39, 40]. SVM uses kernel functions to map the original data to a feature space of higher dimension and then locates an optimal separating hyperplane.
SVR. In comparison with SVM, the objective of SVR is to estimate an unknown continuous valued function y = f(X), which is based on a finite number of samples [41, 42]. The method has been successfully used in many bioinformatics tasks, such as predicting protein B-factors , residue contact numbers , and residue-wise contact orders .
The RF and RFR algorithms were run in the R programming environment (built by the R project, http://www.r-project.org/). To implement SVM and SVR, we used LIBSVM http://www.csie.ntu.edu.tw/~cjlin/ with an RBF kernel. The parameters of SVM or SVR were selected with the LIBSVM parameter selection tool http://www.csie.ntu.edu.tw/~cjlin/libsvmtools/.
Prediction system assessment
where r is Pearson's r, and N, X, and Y are the number of data, experimental ΔΔG value, and predicted ΔΔG value, respectively.
Project name: Prethermut
Project home page: http://www.mobioinfor.cn/prethermut
Operating systems: Linux
Programming language: Perl
Required prerequisite programs: Perl version 5.6 or higher; Foldx 3.0; Modeller v9.7 or higher.
License: GNU General Public License. This license allows the source code to be redistributed and/or modified under the terms of the GNU General Public License, as published by the Free Software Foundation. The source code for the application is available at no charge.
Any restrictions to use by non-academics: None
The authors thank Dr. Vladimir Potapov for providing the S-dataset. We also thank Mr. Xing Qi for revising the paper.
Funding: This work was supported by the National Natural Science Foundation of China (Grant no. 30900839).
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