Tracing Ancestral Specificity of Lectins: Ancestral Sequence Reconstruction Method as a New Approach in Protein Engineering

Abstract

Reconstructing ancestral character states is an exciting but difficult problem. The fossil record carries a great deal of information, but it is incomplete and not always easy to connect to data from modern species. Alternatively, ancestral states can be estimated by modelling trait evolution across a phylogeny, and fitting to values observed in extant species. This approach, however, is heavily dependent on the underlying assumptions, and typically results in wide confidence intervals. An alternative approach is to gain information on ancestral character states from DNA sequence data. This can be done directly when the trait of interest is known to be determined by a single, or a small number, of major effect genes. In some of these cases it can even be possible to investigate an ancestral trait of interest by inferring and resurrecting ancestral sequences in the laboratory. Examples where this has been successfully used to address evolutionary questions range from the nocturnality of early mammals [1], to the loss of functional uricases in primates, leading to high rates of gout, obesity and hypertension in present day humans [2]. Another possibility is to rely on correlations between species traits and the genome average substitution rate/process. For instance, it is well established that the ratio of nonsynonymous to synonymous substitution rate, dN/dS, is generally higher in large than in small species of mammals, presumably due to a reduced

Convergence of Domain Architecture, Structure, and Ligand Affinity in Animal and Plant RNA-Binding Proteins

“…Ancestral sequence resurrection studies have historically relied on an ad-hoc assortment of heuristics to identify particular ancestral nodes for functional analysis, including examining gene duplication patterns or patterns of branch lengths, characterizing changes in selection and projecting functional diversity of extant proteins “back in time” along the phylogeny (Malcolm et al 1990; Shih et al 1993; Ugalde et al 2004; Bridgham et al 2006; Bridgham et al 2009; Zmasek and Godzik 2011; Voordeckers et al 2012; van Hazel et al 2013; Ogawa and Shirai 2014; Whitfield et al 2015; Clifton and Jackson 2016). While these approaches are useful, they are indirect assessments of the hypothesis under examination, which is when and how molecular function has changed across a protein family’s phylogeny.…”

“…Emerging techniques combining ancestral sequence reconstruction (ASR) with laboratory functional assays and structure determination have allowed researchers to meticulously characterize the evolutionary and structural bases for changes in molecular function (Malcolm et al 1990; Shih et al 1993; Ugalde et al 2004; Bridgham et al 2006, 2009; Zmasek and Godzik 2011; Voordeckers et al 2012; van Hazel et al 2013; Ogawa and Shirai 2014; Whitfield et al 2015; Clifton and Jackson 2016). While these approaches provide unprecedented opportunities to rigorously investigate the molecular-functional evolution of protein families (Shih et al 1993; Hanson-Smith et al 2010; Harms and Thornton 2010; Merkl and Sterner 2016), their reliance on detailed experimental methods limits the scale at which ancestral protein resurrection can be applied.…”

Ancestral sequence reconstruction: accounting for structural information by averaging over replacement matrices

“…Other than the valuable evolutionary knowledge gained from these sequences, ancestral proteins may contain desirable properties that modern proteins lack, such as broader substrate range and higher thermostability. Therefore, they can be used as a good starting point for protein engineering (Gumulya and Gillam, 2017;Ogawa and Shirai, 2014).…”