Mega-scale analysis of protein folding stability in biology and protein design

Progress in DNA sequencing and machine learning have illuminated protein sequences and structures on an enormous scale, but protein thermodynamics remains invisible and largely unknown. New approaches are needed to reveal protein folding thermodynamics on the same enormous scale to overcome challenges in biology, medicine, and engineering. We present cDNA display proteolysis, a new method for measuring protein folding stability for up to 900,000 protein domains in a one-week experiment. From 1.7 million measurements in total, we curated a set of ∼800,000 high-quality folding stabilities comprehensively examining single amino acid variants of ∼350 natural and ∼250 de novo designed protein domains 40-72 amino acids in length. Using this immense dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also demonstrated applications of our method and dataset to protein design. The unique scale, speed, and accuracy of cDNA display proteolysis hold the potential to reveal protein folding thermodynamics across the entire universe of protein domains.