Periodic Reporting for period 4 - NoiseRobustEvo (Noise and robustness in the evolution of novel protein phenotypes)
Periodo di rendicontazione: 2022-04-01 al 2023-03-31
Living cells are constantly barraged by perturbations that originate within themselves. Especially abundant are two kinds of such perturbations. The first is gene expression noise, pervasive stochastic variation of transcript and protein levels. The second is mistranslation noise, the misincorporation of amino acids by ribosomes during protein synthesis. Organisms and protein molecules can evolve robustness – the persistence of well-adapted phenotypes – to both kinds of noise. Theory predicts that noise and robustness can affect the adaptive evolution of new proteins, but we do not know whether they help or hinder adaptive evolution. In this project, we studied how noise and robustness affect the evolution of proteins. Specifically, we evolved light-emitting fluorescent proteins towards new color phenotypes via directed laboratory evolution in E. coli, while manipulating noise and robustness. We studied how fast fluorescent proteins evolve new colors and analyzed protein evolutionary dynamics through a combination of high-throughput sequencing, engineering of selected adaptive mutations, and data-driven modeling. Our project showed how a ubiquitous but poorly understood source of phenotypic variation affects protein innovation. It made multiple important discoveries detailed below. These discoveries resolved fundamental open questions in evolutionary biology. In addition, they can help engineers engineer proteins with new functions. Moreover, our work helps to establish fluorescent proteins as a major platform to study the adaptive evolution of protein phenotypes.
We conducted the planned evolution experiments and studied genotypic evolution in all experiments by high-throughput population sequencing. We studied phenotypic evolution through multiple biochemical assays. And we engineered individual protein mutations – for some experiments hundreds of them – in laborious experiments to identify the genetic causes of the phenotypic change we observed during evolution.
Already in preliminary experiments, we made a breakthrough discovery that resolves a long-standing controversy about the question whether evolvability can itself be subject to Darwinian evolution. It proves that the evolvability of a protein can evolve adaptively through DNA mutations that increase its robustness to thermal noise.
We also discovered that noisy gene expression does facilitate the evolution of a new color phenotype. This disproves theory predicting that gene expression noise hinders adaptive evolution. In addition, we found entirely unexpectedly that lowly expressed genes more rapidly evolve new phenotypes than highly expressed.
Our experiments falsified our original hypothesis that mistranslation may accelerate adaptive evolution of a new protein phenotype. However, here too we made an unexpected and important discovery: High mistranslation can prompt the purging of deleterious DNA mutations, and thus helps increase the genetic ‘health’ of an evolving population.
Our experiments also proved that the chaperone GroEL/S does not always buffer deleterious mutations and thus increase robustness to mutations. Instead, it can either buffer deleterious mutations and facilitate their persistence in a population, or potentiate their effect, so that they get purged more quickly from the population.
Other experiments proved that gene duplication increases robustness of genes to mutations, but does not accelerate evolution towards new phenotypes. They falsify a half-century and still hotly debated hypothesis by Susumu Ohno that the increased mutational robustness caused by gene duplication facilitates evolution.
Additional discoveries went beyond the stated goals, and demonstrated new roles for noise and robustness in evolution. One of them is that Darwinian protein evolution can be accelerated by low temperature in a way that is linked to the robustness of proteins to noise. We also demonstrated that correlations between color traits of our study proteins can evolve rapidly. In doing so, they provide the first proof that an important determinant of evolvability may itself evolve.
Multiple of these discoveries are already published in either top generalist journals (Science, Nature Ecology and Evolution) or in top specialist journals (Molecular Biology and Evolution, Genetics). Others are still being prepared for publication in such journals. Both the PI and team members also disseminated results by lecturing at workshops, international conferences, and university seminars.
Already in preliminary experiments, we made a breakthrough discovery that resolves a long-standing controversy about the question whether evolvability can itself be subject to Darwinian evolution. It proves that the evolvability of a protein can evolve adaptively through DNA mutations that increase its robustness to thermal noise.
We also discovered that noisy gene expression does facilitate the evolution of a new color phenotype. This disproves theory predicting that gene expression noise hinders adaptive evolution. In addition, we found entirely unexpectedly that lowly expressed genes more rapidly evolve new phenotypes than highly expressed.
Our experiments falsified our original hypothesis that mistranslation may accelerate adaptive evolution of a new protein phenotype. However, here too we made an unexpected and important discovery: High mistranslation can prompt the purging of deleterious DNA mutations, and thus helps increase the genetic ‘health’ of an evolving population.
Our experiments also proved that the chaperone GroEL/S does not always buffer deleterious mutations and thus increase robustness to mutations. Instead, it can either buffer deleterious mutations and facilitate their persistence in a population, or potentiate their effect, so that they get purged more quickly from the population.
Other experiments proved that gene duplication increases robustness of genes to mutations, but does not accelerate evolution towards new phenotypes. They falsify a half-century and still hotly debated hypothesis by Susumu Ohno that the increased mutational robustness caused by gene duplication facilitates evolution.
Additional discoveries went beyond the stated goals, and demonstrated new roles for noise and robustness in evolution. One of them is that Darwinian protein evolution can be accelerated by low temperature in a way that is linked to the robustness of proteins to noise. We also demonstrated that correlations between color traits of our study proteins can evolve rapidly. In doing so, they provide the first proof that an important determinant of evolvability may itself evolve.
Multiple of these discoveries are already published in either top generalist journals (Science, Nature Ecology and Evolution) or in top specialist journals (Molecular Biology and Evolution, Genetics). Others are still being prepared for publication in such journals. Both the PI and team members also disseminated results by lecturing at workshops, international conferences, and university seminars.
Our work includes several unexpected discoveries that go far beyond state of the art. One of them resolves a decades-old question in evolutionary genetics: Is cryptic variation important for successful Darwinian evolution, and if so, why? The answer is yes, because cryptic variation can reveal unexpected new phenotypes in a new selective environment.
Another breakthrough discovery resolves a long-standing controversy about the question whether evolvability can itself be subject to Darwinian evolution. It proves that the evolvability of a protein can evolve adaptively through DNA mutations that increase its ability to fold, and thus its robustness to thermal noise. In doing so it not only proves that evolvability can evolve, but that it can increase very rapidly through Darwinian evolution.
A further substantial discovery is that Darwinian protein evolution can be accelerated by low temperature, whereas almost all biological processes are accelerated by high temperature. The work is especially important because, first, it is the first experimental study to assess the effect of temperature on phenotypic evolution. Second, it also explains why low temperature accelerates evolution. Mutations that bring forth a new phenotype destabilize evolving proteins, but do so much less strongly at low temperature, because low temperature renders proteins more robust to mutations and to thermal noise, a core subject of the project. Third, the discovery has potentially large practical implications. For example, it shows that low temperature can help protein engineers evolve proteins with new functions, because it helps function-altering mutations to manifest their beneficial effects. It also has implications for how we mitigate the temperature increases caused by global climate change, because high temperature may impede evolutionary rescue – the ability of populations to adapt to otherwise lethal temperature increases.
Another breakthrough discovery resolves a long-standing controversy about the question whether evolvability can itself be subject to Darwinian evolution. It proves that the evolvability of a protein can evolve adaptively through DNA mutations that increase its ability to fold, and thus its robustness to thermal noise. In doing so it not only proves that evolvability can evolve, but that it can increase very rapidly through Darwinian evolution.
A further substantial discovery is that Darwinian protein evolution can be accelerated by low temperature, whereas almost all biological processes are accelerated by high temperature. The work is especially important because, first, it is the first experimental study to assess the effect of temperature on phenotypic evolution. Second, it also explains why low temperature accelerates evolution. Mutations that bring forth a new phenotype destabilize evolving proteins, but do so much less strongly at low temperature, because low temperature renders proteins more robust to mutations and to thermal noise, a core subject of the project. Third, the discovery has potentially large practical implications. For example, it shows that low temperature can help protein engineers evolve proteins with new functions, because it helps function-altering mutations to manifest their beneficial effects. It also has implications for how we mitigate the temperature increases caused by global climate change, because high temperature may impede evolutionary rescue – the ability of populations to adapt to otherwise lethal temperature increases.