Wilson, C. B. | bioRxiv Science

bioRxiv2026

PqiABC forms a membrane-bridging conduit and mediates bidirectional phospholipid transport across the Gram-negative bacterial envelope

The mechanism by which glycerophospholipids are transported between the inner and outer membranes in Gram-negative bacteria remains poorly understood. In Escherichia coli, the paraquat-inducible (Pqi) pathway, comprising the inner membrane protein PqiA, the periplasm-spanning MCE-family protein PqiB, and the outer membrane lipoprotein PqiC, has been implicated in this process. These components are proposed to assemble into a quaternary complex that forms a continuous channel bridging the inner and outer membranes. Here, using neutron reflectometry and quartz crystal microbalance with dissipation monitoring, we perform a dynamic structural analysis of PqiABC within a planar double bilayer membrane-mimetic system. This approach reveals that PqiABC assembles into a stable, envelope-spanning complex anchored to both membranes, consistent with its proposed conduit architecture. Furthermore, using neutron reflectometry in combination with complementary fluorescence-based assays, we demonstrate that PqiABC mediates passive glycerophospholipid transport, supporting bidirectional lipid exchange between membranes. Together, these findings establish PqiABC as a membrane-bridging lipid transport system and provide direct evidence for a mechanism of passive glycerophospholipid equilibration across the bacterial envelope.

biophysics↗

bioRxiv2024

Conformations of a Low-Complexity Protein in Homogeneous and Phase-Separated Frozen Solutions

Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below temperatures TLLPS in the 20-40{degrees} C range. To investigate whether local conformational distributions are detectably different in the homogeneous and phase-separated states of FUS-LC, we performed solid state nuclear magnetic resonance (ssNMR) measurements on solutions that were frozen on sub-millisecond time scales after equilibration at temperatures well above (50{degrees} C) or well below (4{degrees} C) TLLPS. Measurements were performed at 25 K with signal enhancements from dynamic nuclear polarization. Crosspeak patterns in two-dimensional (2D) ssNMR spectra of rapidly frozen solutions in which FUS-LC was uniformly 15N,13C-labeled were found to be nearly identical for the two states. Similar results were obtained for solutions in which FUS-LC was labeled only at Thr, Tyr, and Gly residues, as well as solutions of a FUS construct in which five specific residues were labeled by ligation of synthetic and recombinant fragments. These experiments show that local conformational distributions are nearly the same in the homogeneous and phase-separated solutions, despite the much greater protein concentrations and more abundant intermolecular interactions within phase-separated, protein-rich "droplets". Comparison of the experimental results with simulations of the sensitivity of 2D crosspeak patterns to an enhanced population of {beta}-strand-like conformations suggests that changes in conformational distributions are no larger than 5-10%. Statement of SignificanceLiquid-liquid phase separation (LLPS) in solutions of proteins with intrinsically disordered domains has attracted recent attention because of its relevance to multiple biological processes and its inherent interest from the standpoint of protein biophysics. The high protein concentrations and abundant intermolecular interactions within protein-rich, phase-separated "droplets" suggests that conformational distributions of intrinsically disordered proteins may differ in homogeneous and phase-separated solutions. To investigate whether detectable differences exist, we performed experiments on the low-complexity domain of the FUS protein (FUS-LC) in which FUS-LC solutions were first equilibrated at temperatures well above or well below their LLPS transition temperatures, then rapidly frozen and examined at very low temperatures by solid state nuclear magnetic resonance (ssNMR) spectroscopy. The ssNMR data for homogeneous and phase-separated frozen solutions of FUS-LC were found to be nearly identical, showing that LLPS is not accompanied by substantial changes in the local conformational distributions of this intrinsically disordered protein.

biophysics↗

bioRxiv2022

Triggered functional dynamics of AsLOV2 by time-resolved electron paramagnetic resonance at high magnetic fields

We present time-resolved Gd-Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter-residue distances during a proteins mechanical cycle in the solution state. TiGGER makes use of Gd-sTPATCN as spin labels, whose favorable qualities include a spin-7/2 EPR-active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C-terminus and N-terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light-activated long-range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.

biophysics↗