Final Report Summary - PROTEINARCHEOLOGY (Protein archaeology: reconstructed ancestors for protein engineering and crystallography)
Protein evolution constitutes one of the most appealing topics in life science. However, the major difficulty consists in obtaining experimental data about proteins from past organisms, and for which most (if not all) biomolecules have vanished. However, recent advances in molecular evolution have led to new concepts, like the ancestral reconstruction or ancestral inference. Our aim were to understand how protein evolves, how new functions emerge, and to decipher the selection pressures that yield to functional divergence and / or new functions. We therefore used different approaches and protein models to underline these features, from detailed structural and chemical analysis to advanced techniques in molecular biology.
We explored and developed the ancestral resurrection technique as a powerful engineering tool for improving new catalytic functions, increasing stability and potent crystallisability. We demonstrated in two different protein families that ancestral libraries comprise a far superior engineering method than random mutagenesis libraries to improve new catalytic functions while screening few clones. Indeed, this method allowed the isolation of dozens of active clones with a range of different specificities and higher activities, but screening as few as 200 - 300 variants. Structural studies that we performed demonstrated how ancestral mutations finely tune substrates binding and thus increase catalytic activity or change substrate specificity. As opposed to classical directed evolution experiments where the screening of thousands of clones is required, we show that ancestral libraries can be used in enzyme engineering to improve the catalytic efficiency, evolve a new function including for enzymes where no high throughput screening method is available. This technique has therefore the potential to engineer new enzymatic functions for medical or biotechnological purposes, the generated variants being additionally endowed with increased stability and solubility.
Moreover, we focused on the evolutionary history of proteins that carry an ancient function: the phosphate binding proteins (PBPs). These high affinity proteins that bind phosphate are involved in the key cellular process of phosphate uptake (pst). We have performed a detailed biochemical and structural characterisation of 5 PBPs. We could demonstrate that these ancient proteins are not only under the selection pressure of having a high affinity for phosphate binding, but also for ultra-high selectivity to exclude the chemically close anions such as arsenate. This work yielded the discovery of a unique discrimination mechanism to a level that is unprecedented. The PBPs, being the first proteins shown to be able to discriminate between phosphate and arsenate, can do so because of a unique ultra-specific hydrogen bond that amplifies the small differences between both anions. This discovery might be part of the arsenate resistance mechanism of organisms living in arsenate rich environment, and may have significant importance for the understanding and the design of ultra-specific molecules (e.g. drugs).
We explored and developed the ancestral resurrection technique as a powerful engineering tool for improving new catalytic functions, increasing stability and potent crystallisability. We demonstrated in two different protein families that ancestral libraries comprise a far superior engineering method than random mutagenesis libraries to improve new catalytic functions while screening few clones. Indeed, this method allowed the isolation of dozens of active clones with a range of different specificities and higher activities, but screening as few as 200 - 300 variants. Structural studies that we performed demonstrated how ancestral mutations finely tune substrates binding and thus increase catalytic activity or change substrate specificity. As opposed to classical directed evolution experiments where the screening of thousands of clones is required, we show that ancestral libraries can be used in enzyme engineering to improve the catalytic efficiency, evolve a new function including for enzymes where no high throughput screening method is available. This technique has therefore the potential to engineer new enzymatic functions for medical or biotechnological purposes, the generated variants being additionally endowed with increased stability and solubility.
Moreover, we focused on the evolutionary history of proteins that carry an ancient function: the phosphate binding proteins (PBPs). These high affinity proteins that bind phosphate are involved in the key cellular process of phosphate uptake (pst). We have performed a detailed biochemical and structural characterisation of 5 PBPs. We could demonstrate that these ancient proteins are not only under the selection pressure of having a high affinity for phosphate binding, but also for ultra-high selectivity to exclude the chemically close anions such as arsenate. This work yielded the discovery of a unique discrimination mechanism to a level that is unprecedented. The PBPs, being the first proteins shown to be able to discriminate between phosphate and arsenate, can do so because of a unique ultra-specific hydrogen bond that amplifies the small differences between both anions. This discovery might be part of the arsenate resistance mechanism of organisms living in arsenate rich environment, and may have significant importance for the understanding and the design of ultra-specific molecules (e.g. drugs).