title: Engineering ancestral protein hyperstability.
abstract: Many 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 evolutionarily 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 ∼123°C. 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 of ∼115°C) immediately identifies three mutations that increase the denaturation temperature of LPBCA thioredoxin to ∼128°C. Comparison between evolutionarily 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.
authors.affiliation: Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n, Granada 18071, Spain.
authors.forename: M Luisa
authors.initials: ML
authors.lastname: Romero-Romero
authors.affiliation: Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n, Granada 18071, Spain.
authors.forename: Valeria A
authors.initials: VA
authors.lastname: Risso
authors.affiliation: Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n, Granada 18071, Spain.
authors.forename: Sergio
authors.initials: S
authors.lastname: Martinez-Rodriguez
authors.affiliation: Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n, Granada 18071, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Ibarra-Molero
authors.affiliation: Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n, Granada 18071, Spain.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Sanchez-Ruiz
mesh_terms: D001419:Bacteria / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D057005:Bioengineering / Q000379:methods*; D004795:Enzyme Stability / Q000235:genetics / Q000502:physiology; D019143:Evolution, Molecular; D010802:Phylogeny; D017433:Protein Structure, Secondary; D013879:Thioredoxins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D013879:Thioredoxins
keywords: protein engineering; protein stability; resurrected proteins; thioredoxin