Ancestral sequence reconstruction

Ancestral sequence reconstruction (ASR) – also known as ancestral gene/sequence reconstruction/resurrection – is a technique used in the study of molecular evolution. The method uses related sequences to reconstruct an "ancestral" gene from a multiple sequence alignment. The method can be used to 'resurrect' ancestral proteins and was suggested in 1963 by Linus Pauling and Emile Zuckerkandl. In the case of enzymes, this approach has been called paleoenzymology (British: palaeoenzymology). Some early efforts were made in the 1980s and 1990s, led by the laboratory of Steven A. Benner, showing the potential of this technique. Thanks to the improvement of algorithms and of better sequencing and synthesis techniques, the method was developed further in the early 2000s to allow the resurrection of a greater variety of and much more ancient genes. Over the last decade, ancestral protein resurrection has developed as a strategy to reveal the mechanisms and dynamics of protein evolution. Unlike conventional evolutionary and biochemical approaches to studying proteins, i.e. the so-called horizontal comparison of related protein homologues from different branch ends of the tree of life; ASR probes the statistically inferred ancestral proteins within the nodes of the tree – in a vertical manner (see diagram, right). This approach gives access to protein properties that may have transiently arisen over evolutionary time and has recently been used as a way to infer the potential selection pressures that resulted in present-day sequences. ASR has been used to probe the causative mutation that resulted in a protein's neofunctionalization after duplication by first determining that said mutation was located between ancestors '5' and '4' on the diagram (illustratively) using functional assays. In the field of protein biophysics, ASR has also been used to study the development of a protein's thermodynamic and kinetic landscapes over evolutionary time as well as protein folding pathways by combining many modern day analytical techniques such as HX/MS.