The specific binding of ligands by proteins and the coupling of this process to conformational changes is 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 ATP-binding cassette importer FeuBC, which is involved in bacterial iron uptake. The conformational dynamics of FeuA was assessed via Förster resonance energy transfer, 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 10,000-fold and temporally stabilize the formed conformation 250-fold.

14The specific binding of ligands by proteins and the coupling of this process to conformational changes 15 are fundamental to protein function. We designed a fluorescence-based single-molecule assay and data 16 analysis procedure that allows the simultaneous real-time observation of ligand binding and 17 conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-18 bacillibactin and delivers it to the ABC importer FeuBC, which is involved in iron uptake in bacteria. 19The conformational dynamics of FeuA was assessed via Förster resonance energy transfer (FRET), 20 whereas the presence of the ligand was probed by fluorophore quenching. We reveal that ligand 21 binding shifts the conformational equilibrium of FeuA from an open to a closed conformation. Ligand 22 binding occurs via an induced-fit mechanism, i.e., the ligand binds to the open state and subsequently 23 triggers a rapid closing of the protein. However, FeuA also rarely samples the closed conformation 24 without the involvement of the ligand. This shows that ligand interactions are not required for 25 conformational changes in FeuA. However, ligand interactions accelerate the conformational change 26 10000-fold and temporally stabilize the formed conformation 250-fold. 27 28 29 2 SIGNIFICANCE STATEMENT 30Ligand binding and the coupling of this process to conformational changes in proteins are 31 fundamental to their function. We developed a single-molecule approach that allows the 32 simultaneous observation of ligand binding and conformational changes in the substrate-33 binding protein FeuA. This allows to directly observe the ligand binding process, ligand-34 driven conformational changes as well as rare short-lived conformational transitions that are 35 uncoupled from the ligand. These findings provide insight into the fundamental relation 36 between ligand-protein interactions and conformational changes. Our findings are, however, 37 not only of interest to understand protein function, but the developed data analysis procedure 38 allows the determination of (relative) distance changes in single-molecule FRET experiments, 39for situations in which donor and acceptor fluorophore are influenced by quenching processes. 40 41 42 48 the energy landscape that connect unliganded and liganded conformational states (Fig. 1). In the 49 induced-fit mechanism, ligand interactions trigger conformational changes, whereas in the 50 conformational selection mechanism, ligand interactions selectively stabilize a subset of 51 conformations that are already present in the unliganded protein ( Fig. 1). Both mechanisms require 52 intermediate states that are formed during the ligand-binding process. For example, when a protein 53 switches between two conformational states, such as an open and closed conformation ( Fig. 1), an 54 open-liganded state in the induced-fit mechanism or a closed-unliganded state in the conformational 55 3 selection mechanism, are essential intermediate states. However, the study of such transient and 56 thermody...

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 remain poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the periplasmic-binding protein–like II domain family, have undergone divergent evolution, leading to adaptation of their structural dynamics. We performed a structural analysis on ∼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 Förster resonance energy transfer and Hydrogen–Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enable distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extracytoplasmic transport-related proteins. Structural embellishments of the core created 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 evolution and are applicable to a large number of domain architectures.