Modulation of E.Coli Dihydrofolate Reductase (DHFR) Activity by Site-Directed Chemical Cross-Linking (SDXL) of the M20 and FG Loops

Abstract

Generally, proteins are comprised of highly complex structures and fluctuations of local or global character are believed to play critical roles in protein function. In the case of E.Coli dihydrofolate reductase (DHFR), previous structural studies have already revealed some aspects of DHFR's conformational flexibility. Among the most prominent displays of flexibility are motions between loops M20 and FG. We have employed site-directed chemical cross-linking (SDXL) to modulate loop motions and by extension modulate activity, flexibility, stability and substrate affinity. Toward this end, we have utilized both computational and experimental approaches. Target residues were identified by average distance matrices and principle component analysis calculated through molecular dynamic (MD) simulations at various temperatures. Activity measurements for a mirrored pair of cross-linked residues resulted in reduced activity for shorter cross-links, while longer cross-links returned the activity to near unmodified protein levels. Mass spectrometry was used to determine the effects on stability against thermal and chaotropic denaturation by observing changes in the charge state envelope associated with protein unfolding. Hydrogen/deuterium exchange (HDX) combined with mass spectrometry was used to probe local conformational changes both proximal and distal to the cross-link site. Finally with the ability to observe both free and bound species, mass spectrometry was able to monitor changes in substrate affinity as correlated with cross-link length. With this work we have shown that SDXL is able to modulate enzymatic activity and stability by controlling motions associated with thermal fluctuations.