The most probable ancestral sequence reconstruction yields proteins without systematic bias in thermal stability or activity

Abstract

Ancestral sequence resurrection (ASR) is the inference of extinct biological sequences from extant sequences, the most popular of which are based on probabilistic models of evolution. ASR is becoming a popular method for studying the evolution of enzyme characteristics. The properties of ancestral enzymes are biochemically and biophysically characterized to gain some knowledge regarding the origin of some enzyme property. Current methodology relies on resurrection of the single most probable (SMP) sequence and is systematically biased. Previous theoretical work suggests this will result in a thermostability bias in resurrected SMP sequences, and even the activity, calling into question inferences derived from ancestral protein properties. We experimentally test the potential stability bias hypothesis by resurrecting 40 malate and lactate dehydrogenases. Despite the methodological bias in resurrecting an SMP protein, the measured biophysical and biochemical properties of the SMP protein are not biased in comparison to other, less probable, resurrections.