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Inferring stabilizing mutations from protein phylogenies: application to influenza hemagglutinin.

Bloom JD, Glassman MJ - PLoS Comput. Biol. (2009)

Bottom Line: We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny.This approach is able to predict published experimentally measured mutational stability effects (DeltaDeltaG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach.Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California, USA. jesse.bloom@gmail.com

ABSTRACT
One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (DeltaDeltaG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution.

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Plaque assays of wildtype, temperature-sensitive (ts), and tsinfluenza with predicted stabilizing hemagglutinin mutations.All four of the single mutations allow the virus to plaque at highertemperatures than the ts parent. The multiple mutants plaque moreeffectively at higher temperatures than the single mutants. Mutationsare named according to the numbering scheme described in Table 1.
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pcbi-1000349-g011: Plaque assays of wildtype, temperature-sensitive (ts), and tsinfluenza with predicted stabilizing hemagglutinin mutations.All four of the single mutations allow the virus to plaque at highertemperatures than the ts parent. The multiple mutants plaque moreeffectively at higher temperatures than the single mutants. Mutationsare named according to the numbering scheme described in Table 1.

Mentions: To confirm the increased temperature stability of viruses carrying the fourapparently stabilizing mutations, we re-grew the viruses from the encodingplasmids and again plaqued them at various temperatures that now included38.0°C. The results of these plaque assays are shown in Figure 11. All four mutantswere clearly more thermotolerant than their temperature-sensitive parent,although still less so than the wildtype virus. To test whether the stabilizingmutations had cumulative effects, we constructed a double-mutant carrying two ofthe stabilizing mutations, and a triple-mutant carrying three of the stabilizingmutations. As can be seen in Figure11, these multiple mutants were more thermotolerant than the singlemutants, as indicated by better plaquing at 38.5°C.


Inferring stabilizing mutations from protein phylogenies: application to influenza hemagglutinin.

Bloom JD, Glassman MJ - PLoS Comput. Biol. (2009)

Plaque assays of wildtype, temperature-sensitive (ts), and tsinfluenza with predicted stabilizing hemagglutinin mutations.All four of the single mutations allow the virus to plaque at highertemperatures than the ts parent. The multiple mutants plaque moreeffectively at higher temperatures than the single mutants. Mutationsare named according to the numbering scheme described in Table 1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664478&req=5

pcbi-1000349-g011: Plaque assays of wildtype, temperature-sensitive (ts), and tsinfluenza with predicted stabilizing hemagglutinin mutations.All four of the single mutations allow the virus to plaque at highertemperatures than the ts parent. The multiple mutants plaque moreeffectively at higher temperatures than the single mutants. Mutationsare named according to the numbering scheme described in Table 1.
Mentions: To confirm the increased temperature stability of viruses carrying the fourapparently stabilizing mutations, we re-grew the viruses from the encodingplasmids and again plaqued them at various temperatures that now included38.0°C. The results of these plaque assays are shown in Figure 11. All four mutantswere clearly more thermotolerant than their temperature-sensitive parent,although still less so than the wildtype virus. To test whether the stabilizingmutations had cumulative effects, we constructed a double-mutant carrying two ofthe stabilizing mutations, and a triple-mutant carrying three of the stabilizingmutations. As can be seen in Figure11, these multiple mutants were more thermotolerant than the singlemutants, as indicated by better plaquing at 38.5°C.

Bottom Line: We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny.This approach is able to predict published experimentally measured mutational stability effects (DeltaDeltaG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach.Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California, USA. jesse.bloom@gmail.com

ABSTRACT
One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (DeltaDeltaG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution.

Show MeSH
Related in: MedlinePlus