The Evolution of the S100A4 Ensemble

Abstract

Understanding the evolution of proteins is essential for interpreting the complex mechanisms driving biological systems. The idea of intramolecular epistasis, which outlines a situation in which the effects of one mutation in a protein depend on the existence of other mutations in the same protein, is central to this investigation. Proteins’ evolutionary paths are shaped by these complex interactions, which also affect the variety and adaptability of their functions. Proteins display an intriguing variety of epistatic patterns, ranging from long-range interactions between far-off sites to dynamic changes triggered by their environments. Evolutionary biochemistry has struggled for decades to provide thorough predictive models for these intricate patterns. Instead of treating proteins as static structures, this study views them as dynamic entities with a variety of conformations. With a focus on human S100A4 and its ancestral counterparts, AncA4 and AncA2A4, this investigation seeks to shed light on the complex dynamics of ensemble epistasis and its evolutionary implications over time scales. Urea unfolding experiments for S100A4, AncA2A4, and AncA4 quantified free energy. Expected alignment of ΔG values across signals within each protein was not observed, indicating potential oligomerization taking place during unfolding. Observed variations in ΔG values during unfolding between these three proteins suggest potential influences of evolutionary mutations. Additionally, EDTA titrations for calcium binding demonstrated unexpected patterns, indicating a more intricate cooperativity in calcium binding dynamics within the protein ensemble. Nevertheless, a two-state system for binding calcium was observed, where low affinity binding took place first, followed by high affinity binding.