Abstract
If the fitness effects of amino acid mutations are conditional on genetic background, then mutations can have different effects depending on the sequential order in which they occur during evolutionary transitions in protein function. A key question concerns the fraction of possible mutational pathways connecting alternative functional states that involve transient reductions in fitness. Here we examine the functional effects of multiple amino acid substitutions that contributed to an evolutionary transition in the oxygenation properties of avian hemoglobin (Hb). The set of causative changes included mutations at intradimer interfaces of the Hb tetramer. Replacements at such sites may be especially likely to have epistatic effects on Hb function since residues at intersubunit interfaces are enmeshed in networks of salt bridges and hydrogen bonds between like and unlike subunits; mutational reconfigurations of these atomic contacts can affect allosteric transitions in quaternary structure and the propensity for tetramer–dimer dissociation. We used ancestral protein resurrection in conjunction with a combinatorial protein engineering approach to synthesize genotypes representing the complete set of mutational intermediates in all possible forward pathways that connect functionally distinct ancestral and descendent genotypes. The experiments revealed that 1/2 of all possible forward pathways included mutational intermediates with aberrant functional properties because particular combinations of mutations promoted tetramer–dimer dissociation. The subset of mutational pathways with unstable intermediates may be selectively inaccessible, representing evolutionary roads not taken. The experimental results also demonstrate how epistasis for particular functional properties of proteins may be mediated indirectly by mutational effects on quaternary structural stability.
Highlights
When an evolutionary transition in protein function involves multiple mutational steps, a number of important questions can be addressed by experimentally examining the full set of possible intermediate genotypes that connect the ancestral starting point and the evolved endpoint (Weinreich 2010; Weinreich, et al 2013; de Visser and Krug 2014; Hartl 2014)
If n mutations are associated with an evolved change in protein function, and if a binary combination of ancestral/ derived amino acids are possible at each site, the key question is whether all n mutations have either neutral or beneficial effects in each of the 2n possible multi-allelic combinations
We focus on one particular species, Tschudi’s nightjar (Hydropsalis decussata), a strictly lowland bird that has evolved a reduced Hb-O2 affinity relative to its highland sister species, the band-winged nightjar (Hydropsalis longirostris), which occurs at elevations ranging from $2000 m to 4400 m above sea level in the Andes (Schulenberg et al 2007; Benham et al 2011)
Summary
When an evolutionary transition in protein function involves multiple mutational steps, a number of important questions can be addressed by experimentally examining the full set of possible intermediate genotypes that connect the ancestral starting point and the evolved endpoint (Weinreich 2010; Weinreich, et al 2013; de Visser and Krug 2014; Hartl 2014). Amino acid replacements at intradimeric contacts can affect the allosteric transition in quaternary structure and the propensity for tetramer–dimer dissociation (Fronticelli et al 1994; Tsuruga et al 1998; Vasquez et al 1999; Chang et al 2002; Yasuda et al 2002; Shikama and Matsuoka 2003; Bellelli et al 2006; Natarajan et al 2013) Such replacements may be especially likely to have epistatic effects on Hb function since residues at intersubunit interfaces are involved in networks of salt bridges and hydrogen bonds with multiple residues in like and unlike subunits. We assessed the fraction of mutational pathways connecting functionally distinct ancestral and descendant genotypes that include potentially deleterious intermediate steps
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