A number of experimental analyses and proposed scenarios support that ancient life was thermophilic. In congruence with this hypothesis, proteins encoded by reconstructed sequences corresponding to ancient phylogenetic nodes often display very high stability. Here we show that such "reconstructed ancestral hyperstability" can be further engineered on the basis of a straightforward approach that uses exclusively information afforded by the ancestral reconstruction process itself. Since evolution does not imply continuous progression, screening of the mutations between two evolutionary related resurrected ancestral proteins may identify mutations that further stabilize the most stable one. To explore this approach, we have used a resurrected thioredoxin corresponding to the last common ancestor of the cyanobacterial, deinococcus and thermus groups (LPBCA thioredoxin), which has a denaturation temperature of about 123 ºC. This high value is within the top 0.1% of the denaturation temperatures in the ProTherm database and, therefore, achieving further stabilization appears a priori as a challenging task. Nevertheless, experimental comparison with a resurrected thioredoxin corresponding to the last common ancestor of bacteria (denaturation temperature about 115 ºC) immediately identifies 3 mutations that increase the denaturation temperature of LPBCA thioredoxin to about 128 ºC. Comparison between evolutionary related resurrected ancestral proteins thus emerges as a simple approach to expand the capability of ancestral reconstruction to search sequence space for extreme protein properties of biotechnological interest. The fact that ancestral sequences for many phylogenetic nodes can be derived from a single alignment of modern sequences should contribute to the general applicability of this approach.
- protein engineering
- resurrected proteins
- protein stability
- ©2016 The Author(s)
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