Resurrecting prehistoric parvalbumins to explore the evolution of thermal compensation in extant Antarctic fish parvalbumins

Abstract

SummaryParvalbumins (PVs) from Antarctic notothenioid fishes display a pattern of thermal adaptation that likely reflects evolutionary changes in protein conformational flexibility. We have used ancestral sequence reconstruction and homology modeling to identify two amino acid changes that could potentially account for the present thermal sensitivity pattern of Antarctic fish PVs as compared to a PV from a theoretical warm-adapted ancestral fish. To test this hypothesis, ancient PVs were resurrected in the lab using PV from the notothenioid Gobionotothen gibberifrons as a platform for introducing mutations comparable to the reconstructed ancestral PV sequences. The wild-type PV (WT) and three mutant expression constructs were engineered: lysine 8 to asparagine (K8N), lysine 26 to asparagine (K26N) and a double mutant (DM). Calcium equilibrium dissociation constants (Kd) vs. temperature curves for all mutants were right-shifted, as predicted, relative to that of WT PV. The Kd values for the K8N and K26N single mutants were virtually identical at all temperatures and showed an intermediate level of thermal sensitivity. The DM construct displayed a full conversion of thermal sensitivity pattern to that of a PV from a warm/temperate adapted fish. Additionally, the Kd vs temperature curve for the WT construct revealed greater thermal sensitivity as compared to the mutant constructs. Measurements of the rates of Ca2+ dissociation (koff) showed that all mutants generally had slower koff values than WT at all temperatures. Calculated rates of Ca2+ binding (kon) for the K8N and K26N mutants were similar to values for the WT PV at all temperatures. In contrast, the calculated kon values for the DM PV were faster, providing mechanistic insights into the nature of potentially adaptive changes in Ca2+ binding in this PV. The overall results suggest that the current thermal phenotype of Antarctic PVs can be recapitulated by just two amino acid substitutions.