Muthahari, Y. | Search bioRxiv

bioRxiv2020

Evolution of structural dynamics in bilobed proteins

Novel biophysical tools allow the structural dynamics of proteins, and the regulation of such dynamics by binding partners, to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remains poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the type-II periplasmic binding protein domain family, have undergone divergent evolution leading to modification of their structural dynamics and function. We performed a structural analysis of ~600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Forster resonance energy transfer and Hydrogen-Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enables distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extra-cytoplasmic transport-related proteins. Structural embellishments of the core created new interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately, altered substrate specificity. Our findings reveal an as yet unrecognized mechanism for the emergence of functional promiscuity during long periods of protein evolution and are applicable to a large number of domain architectures.

biophysics↗

bioRxiv2019

Single-molecule observation of ligand binding and conformational changes in FeuA

The specific binding of ligands by proteins and the coupling of this process to conformational changes are fundamental to protein function. We designed a fluorescence-based single-molecule assay and data analysis procedure that allows the simultaneous real-time observation of ligand binding and conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-bacillibactin and delivers it to the ABC importer FeuBC, which is involved in iron uptake in bacteria. The conformational dynamics of FeuA was assessed via Forster resonance energy transfer (FRET), whereas the presence of the ligand was probed by fluorophore quenching. We reveal that ligand binding shifts the conformational equilibrium of FeuA from an open to a closed conformation. Ligand binding occurs via an induced-fit mechanism, i.e., the ligand binds to the open state and subsequently triggers a rapid closing of the protein. However, FeuA also rarely samples the closed conformation without the involvement of the ligand. This shows that ligand interactions are not required for conformational changes in FeuA. However, ligand interactions accelerate the conformational change 10000-fold and temporally stabilize the formed conformation 250-fold.\n\nSIGNIFICANCE STATEMENTLigand binding and the coupling of this process to conformational changes in proteins are fundamental to their function. We developed a single-molecule approach that allows the simultaneous observation of ligand binding and conformational changes in the substrate-binding protein FeuA. This allows to directly observe the ligand binding process, ligand-driven conformational changes as well as rare short-lived conformational transitions that are uncoupled from the ligand. These findings provide insight into the fundamental relation between ligand-protein interactions and conformational changes. Our findings are, however, not only of interest to understand protein function, but the developed data analysis procedure allows the determination of (relative) distance changes in single-molecule FRET experiments, for situations in which donor and acceptor fluorophore are influenced by quenching processes.

biophysics↗