Biology subjects

Furlong, E. J.

Publications and source records attributed to Furlong, E. J..

4 recordsLinked to original sources

bioRxiv2026

The Lower γ Region Ensures Unidirectional Rotation and Torque Generation in the Latter Half of the 80° Substep of F1-ATPase

F1-ATPase achieves unidirectional rotation of its {gamma} shaft through coordinated conformational cycling of the 3{beta}3 ring, yet how the shaft itself enforces directionality remains unclear. Here we analyzed an axle-less TF1 lacking the lower half of the rotor shaft by combining single-molecule rotation assays with cryo-EM structural analysis under catalysis conditions. Under ATP-saturated conditions, wild-type TF1 exhibited only three pauses per turn corresponding to the catalytic dwells, whereas axle-less TF1 exhibited six pauses per turn, indicating the presence of an additional intermediate during rotation. This previously unreported intermediate dwell was observed at 40{degrees} between the binding and catalytic dwells. High-speed recordings revealed frequent backsteps confined to the 40{degrees} transition between the intermediate and catalytic dwells. Analysis of the dwell-time distributions indicated an approximately zero free energy bias between these two states, consistent with thermally driven interconversion. Cryo-EM resolved three corresponding intermediates--binding, intermediate, and catalytic dwells--showing a major {beta} conformational change from 0{degrees} to 40{degrees}, but minimal {beta} rearrangement between 40{degrees} and 80{degrees}, suggesting that the {beta} power stroke is not operative in this interval. Analysis of the {gamma} rotational scheme indicates that the driving force changes across the 0-80{degrees} step: the 0-40{degrees} advance proceeds via a power stroke to an intermediate dwell, whereas the subsequent 40{degrees} transition to the catalytic dwell lacks a power stroke and is dominated by thermal fluctuations. Taken together, these findings suggest that the lower {gamma} region contributes to unidirectional rotation and torque generation specifically in the latter half of the 0-80{degrees} step, while the initial half can proceed even without contributions from the lower {gamma} region.

biophysics↗

bioRxiv2025

Molecular structure and nickel binding capacity of Proteus mirabilis UreE

UreE is a nickel chaperone required for the safe and efficient delivery of nickel to the active site of the metalloenzyme, urease; a key virulence factor of the urinary tract pathogen, Proteus mirabilis. We investigated the structural features of P. mirabilis UreE using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma-mass spectrometry. Here, we report a 2.0 [A] crystal structure of homodimeric PmUreE and show it has capacity to bind five nickel ions per dimer. Truncation of the histidine-rich C-terminus reduced nickel binding capacity by two nickel ions per dimer and comparison with homologous UreE structures allowed the assignment of putative nickel binding sites within the PmUreE structure. These findings increase our understanding of how PmUreE binds nickel and ultimately prevents this toxic metal from causing significant cellular damage in P. mirabilis.

biochemistry↗

bioRxiv2024

The molecular structure of an axle-less F1-ATPase

F1Fo ATP synthase is a molecular rotary motor that can generate ATP using a transmembrane proton motive force. Isolated F1-ATPase catalytic cores can hydrolyse ATP, passing through a series of conformational states involving rotation of the central {gamma} rotor subunit and the opening and closing of the catalytic {beta} subunits. Cooperativity in F1-ATPase has long thought to be conferred through the {gamma} subunit, with three key interaction sites between the {gamma} and {beta} subunits being identified. Single molecule studies have demonstrated that the F1 complexes lacking the {gamma} axle still "rotate" and hydrolyse ATP, but with less efficiency. We solved the cryogenic electron microscopy structure of an axle-less Bacillus sp. PS3 F1-ATPase. The unexpected binding-dwell conformation of the structure in combination with the observed lack of interactions between the axle-less {gamma} and the open {beta} subunit suggests that the complete {gamma} subunit is important for coordinating efficient ATP binding of F1-ATPase.

biochemistry↗

bioRxiv2018

Engineered variants provide new insight into the structural properties important for activity of the highly dynamic, trimeric protein disulfide isomerase, PmScsC

Suppressor of copper sensitivity protein C from Proteus mirabilis (PmScsC) is a homotrimeric disulfide isomerase that plays a role in copper tolerance - a key virulence trait of the uropathogen. Each protomer of the enzyme has an N-terminal trimerisation stem (59 residues) containing a flexible linker (11 residues) connected to a thioredoxin-fold-containing catalytic domain (163 residues). Here, we characterise two PmScsC variants, PmScsC{Delta}N and PmScsC{Delta}Linker. PmScsC{Delta}N, is an N-terminally truncated form of the protomer with two helices of the trimerisation stem removed, generating a protein with dithiol oxidase rather than disulfide isomerase activity. The crystal structure of PmScsC{Delta}N reported here reveals - as expected - a monomer that is structurally similar to the catalytic domain of native PmScsC. The second variant PmScsC{Delta}Linker was designed to remove the 11 amino acid linker and we show that it generates a protein that has neither disulfide isomerase nor dithiol oxidase activity. The crystal structure of PmScsC{Delta}Linker reveals a trimeric arrangement, with the catalytic domains packed together very closely. Small angle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentration, whereas PmScsC{Delta}Linker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations. These findings increase our understanding of disulfide isomerase activity, showing how (i) oligomerisation, (ii) spacing between, and (iii) dynamic motion of, catalytic domains in PmScsC all contribute to its native function.

biochemistry↗