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

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. In addition, the SMP protein property seems to be representative of the ancestral probability distribution. Therefore, the conclusions and inferences drawn from the SMP protein are likely not a source of bias. Significance Ancestral sequence resurrection (ASR) is a powerful tool for: determining how new protein functions evolve; inferring the properties of an environment in which species existed; and protein engineering applications. We demonstrate, using lactate and malate dehydrogenases (L/MDHs), that resurrecting the single most probable sequence (SMP) from a maximum likelihood phylogeny does not result in biased activity and stability relative to sequences sampled from the posterior probability distribution. Previous studies using experimentally measured phenotypes of SMP sequences to make inferences about the environmental conditions and the path of evolution are likely not biased in their conclusions. Serendipitously, we discover ASR is also a valid tool for protein engineering because sampled reconstructions are both highly active and stable. ### Competing Interest Statement The authors have declared no competing interest.