Epistasis

Not all high-fitness sequences have plausible evolutionary paths from lower-fitness starting points via sequential introduction of mutations

I first saw this precise idea articulated by Weinreich et al. in their aptly named "Darwinian evolution can follow only very few mutational paths to fitter proteins"[1]. Many mutation combinations don't have additive effects on every aspect governing protein fitness (expression, stability, function, etc.), and

Antibody-antigen binding

Conformational entropy could still matter in miniprotein binder design

Antibody V-regions improve their affinity for targets by both creating more high-energy interactions and reducing the conformational entropy of their antigen-binding loops[1][2]. Entropy's importance in antibody-antigen affinity seems obvious, given that loop residues largely mediate binding[3][4]. But for de novo-designed miniprotein binders, which often

Epistasis

Antibodies show evidence of significant epistasis in biophysical properties but not binding

Antibodies undergo somatic mutation during affinity maturation in ways that show evidence of epistasis in biophysical properties like expression and polyspecificity, but not binding [1][2]. In a study by Kirby et al 2025[1:1] on 12 anti-SARS-CoV-2 antibodies using deep mutational scanning and yeast display, no combination of