Protein sequence influence on single-molecule transport dynamics

ATP-Binding Cassette (ABC) Transporters are paradigmatic molecular machines that use ATP binding and hydrolysis to power transmembrane transport of diverse substrates. Knowledge of their mechanism comes primarily from static structures of stable intermediates along the transport cycle. We recently developed a single- molecule fluorescence resonance energy transfer (smFRET) system that exploits quenching by vitamin B12 to track its physical movement in real time during ATP-driven transport by nanodisc-reconstituted E. coli BtuCD-F, a mechanistically enigmatic type II ABC importer. Our smFRET quenching (smFRET-Q) measurements demonstrate that BtuCD-F employs a novel mechanism in which transmembrane translocation of B12 is driven by high-energy intermediate conformations that have short, sub-second lifetimes making them inaccessible to standard structural methods. The first conformational change, which is dynamically driven by hydrolysis of a single ATP molecule, moves B12 from its periplasmic binding site into the transmembrane domain, while the second, which is driven by the formation of a hyper-stable complex between the BtuCD and BtuF subunits, delivers B12 to the cytoplasm. Hydrolysis of a second single ATP molecule then dynamically drives dissociation of BtuCD from BtuF to restore the transporter to the starting conformation. One novel feature of this mechanism is that the homodimeric ATPase and transmembrane domains progress through a fundamentally asymmetrical conformational reaction cycle in which the powerstroke is driven by ATP hydrolysis rather than ATP binding. In contrast, previously characterized superfamily members employ a two-fold symmetrical conformational reaction cycle in which the powerstroke is driven by cooperative ATP binding, while ATP hydrolysis gates relaxation back to the conformational ground-state. Our smFRET-Q methods have thus provided unprecedented information on the mechanism of a paradigmatic transport process while visualizing translocation of single substrate molecules in real time. We now propose to harness their high functional and temporal resolution to elucidate how the protein sequence controls the key conformational changes dynamically driving B12 transport by BtuCD-F. We will use double mutant-cycle analyses to investigate how packing interactions in the transmembrane domains control the conformational landscape and furthermore how local electrostatic interactions in the ATPase active site are propagated into directional progression through functional trajectories in that landscape. These studies will be guided by advanced protein primary sequence analysis methods including analyses of evolutionary correlations between pairs of amino acids. The high resolution of the methods we will use to characterize the mutant proteins will elucidate the structural mechanism by which ATP hydrolysis by a paradigmatic protein nanomachine controls functional protein dynamics to efficiently perform mechanical work on a molecular size scale. These studies will moreover provide insight into how protein dynamics constrains protein evolution while simultaneously testing methods to harness cutting-edge sequence-analysis methods to gain insight into functional protein dynamics.