Zhao, V. Y. | bioRxiv Science

bioRxiv2022

Systematic conformation-to-phenotype mapping via limited deep-sequencing of proteins

Non-native conformations drive protein misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well-suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.

biophysics↗

bioRxiv2021

Switching an active site helix in dihydrofolate reductase reveals limits to sub-domain modularity

To what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study sub-domain modularity by creating 20 chimeras of an enzyme, E. coli dihydrofolate reductase (DHFR), in which a catalytically important, 10-residue -helical sequence is replaced by -helical sequences from a diverse set of proteins. The chimeras stably fold but have a range of diminished thermal stabilities and catalytic activities. Evolutionary coupling analysis indicates that the residues of this -helix are under selection pressure to maintain catalytic activity in DHFR. We performed molecular dynamics simulations using replica exchange with solute-tempering. Chimeras with low catalytic activity exhibit non-helical conformations that block the binding site and disrupt the positioning of the catalytically essential residue D27. Simulation observables and in vitro measurements of thermal stability and substrate binding affinity are strongly correlated. Several E. coli strains with chromosomally integrated chimeric DHFRs can grow, with growth rates that follow predictions from a kinetic flux model that depends on the intracellular abundance and catalytic activity of DHFR. Our findings show that although -helices are not universally substitutable, the molecular and fitness effects of modular segments can be predicted by the biophysical compatibility of the replacement segment. Statement of Significance-helices are ubiquitous components of protein structure that exhibit a degree of independent folding behavior, making them plausible structural modules within proteins. Here, we assess the effects of switching the sequence of an -helix in an essential enzyme for -helical sequences from evolutionarily unrelated proteins. The resultant chimeric proteins can still fold but enzymatic activity, stability, and cellular growth rates are negatively affected. Computational investigations reveal how residues in an -helix have been shaped by selection pressure to maintain catalytic activity and a specific, helical conformation of the protein. More broadly, we illustrate how molecular and fitness effects of switching protein segments depend on the protein and cellular context.

biophysics↗