@article {zhao2021switching, title = {Switching an Active Site Helix in Dihydrofolate Reductase Reveals Limits to Subdomain Modularity}, journal = {Biophysical Journal}, year = {2021}, publisher = {Elsevier}, abstract = {To what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study subdomain modularity by creating 20 chimeras of an enzyme,\ Escherichia colidihydrofolate reductase (DHFR), in which a catalytically important, 10-residue\ α-helical sequence is replaced by\ α-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\ α-helix are under selection pressure to maintain catalytic activity in DHFR. Reversion to phenylalanine at key position 31 was found to partially restore catalytic activity, which could be explained by evolutionary coupling values. We performed molecular dynamics simulations using replica exchange with solute tempering. Chimeras with low catalytic activity exhibit nonhelical 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\ α-helices are not universally substitutable, the molecular and fitness effects of modular segments can be predicted by the biophysical compatibility of the replacement segment.}, issn = {0006-3495}, doi = {10.1016/j.bpj.2021.09.032}, url = {https://www.cell.com/biophysj/fulltext/S0006-3495(21)00794-3}, author = {Zhao, Victor Y. and Rodrigues, Jo{\~a}o V. and Elena R. Lozovsky and Daniel L. Hartl and Shakhnovich, EugeneI.} }