Understanding how protein structure and function are determined by amino acid sequence is fundamental to biochemistry. Evolutionary biochemistry extends the classic sequence–structure–function paradigm by incorporating evolutionary history to understand how amino acid changes in the deep past gave rise to the structures and functions we see in modern proteins. However, which specific residues drove the emergence of protein structures and functions remains poorly understood. In this thesis, I apply evolutionary biochemistry approaches to address two central questions: What amino acid changes gave rise to multimerization and allostery, and how is allostery maintained as sequences diverge over evolutionary time? Using hemoglobin, an α₂β₂ heterotetramer whose oxygen transport is regulated by allosteric effectors, I investigate the origins of multimerization and allostery. Using ancestral sequence reconstruction and biochemical experiments, I show that modern hemoglobin’s quaternary structure and allosteric regulation arose through a small number of substitutions in an ancestral homodimer: two substitutions established the heterotetramer, and four additional changes were sufficient to confer allosteric regulation for oxygen binding. This simplicity is made possible by intrinsic features of hemoglobin: symmetry within the multimer amplifies the effects of interface mutations, while an ancient helix movement underlies the conformational transitions required for allostery. These results demonstrate how new, complex protein functions can evolve through simple genetic changes that exploit ancestral features. To examine how allostery is maintained as sequences diverge over evolutionary time, I studied the tetracycline repressor protein family, a group of allosteric proteins that switch between DNA-bound repressor and derepressor states upon effector binding. Through phylogenetics, ancestral sequence reconstruction, and deep mutational scanning, I show that the effect of residue changes on allosteric function shift continuously across evolutionary history. These effects are shaped by interactions with other residues distributed throughout the protein and have been changing since the last common ancestor of proteobacteria. These results demonstrate that allostery depends on amino acids whose effects are highly sensitive to changes at other sites, and that the genetic basis of allostery evolves persistently over time. Together, this work reveals that the emergence of multimerization and allostery can arise through surprisingly simple residue changes, while the maintenance of allostery over time is shaped by pervasive historical contingency.