Type IV secretion systems (T4SSs) are multiprotein nanomachines that mediate conjugative DNA transfer and contribute to the dissemination of antimicrobial resistance genes. The IncHI1 plasmid R27 encodes TrhW, a periplasmic T4SS-associated protein homologous to the F-plasmid proteins TraW and TrbC, which are expressed as separate polypeptides in many F-like systems but are fused into a single protein in IncHI1 plasmids. Despite its proposed role in conjugation, the structural organization of TrhW has remained unknown. Here, we employed complementary computational and solution-based biophysical approaches to characterize the oligomeric state, architecture, and conformational properties of TrhW. Size-exclusion chromatography (SEC) coupled with multi-angle light scattering demonstrated that TrhW exists as a monomer in solution, while circular dichroism spectroscopy revealed a structured protein containing substantial non-canonical secondary structure consistent with flexible regions. SEC-SAXS (small-angle x-ray scattering) showed that TrhW adopts an elongated, non-globular architecture with a radius of gyration of 44.6 Å and a maximum particle dimension of 160 Å. Ab initio reconstructions, BilboMD, and ensemble optimization modeling demonstrated that the experimental scattering data are best described by an ensemble of interconverting conformations rather than a single static structure. Although AlphaFold accurately predicts the overall domain organization of TrhW, the solution-state analyses reveal substantial conformational heterogeneity arising from both the N-terminal region and inter-domain motion. Together, these findings establish TrhW as a dynamic multidomain protein and provide the first experimental structural characterization of this conserved T4SS component, offering a framework for future studies investigating its role in IncHI1 conjugation.
We measure the picosecond dynamics of labyrinthine stripe domains with chiral Néel-type domain walls using time-resolved x-ray resonant magnetic scattering (XRMS). At the stripe-domain wavevector, the helicity-summed signal shows ultrafast demagnetization and recovery together with a weak oscillatory contribution attributed to laser-launched coherent surface phonons. In contrast, the dichroic signal, which is sensitive to the in-plane magnetization within the Néel domain walls, exhibits a pronounced oscillatory response. Interestingly, the observed frequency of domain wall dynamics decreases with increasing pump fluence. We associate this frequency decrease with a transient softening of the domain-wall restoring potential following optical excitation. This finding highlights that time-resolved XRMS isolates internal domain-wall dynamics and provides reciprocal-space access to wall-specific degrees of freedom in disordered stripe textures on picosecond time scales.
John L. Markley was an early pioneer of the application of nuclear magnetic resonance (NMR) spectroscopy to investigate the structure, dynamics, and function of biological macromolecules by NMR. A recurring theme of his career is the development of standards and shared resources that have enabled the biological NMR community to coherently and collectively advance the disciplines of structural biology and metabolomics. Spanning development standards for atom nomenclature, founding shared NMR resources at Purdue and UW-Madison, and founding the Biological Magnetic Resonance Data Bank (BMRB) and shepherding BMRB membership in the Worldwide Protein Data Bank, this appreciation revisits the many and lasting contributions by our friend and colleague John Markley in support of the global structural biology community.
The SARS-CoV-2 virus causes COVID-19, and several of its gene products have been successfully targeted for antiviral drug development, including the 3C-like protease (3CLpro). The substrate-binding site of 3CLpro exhibits significant conformational plasticity. While available X-ray crystal structures reveal substantial loop variability and molecular dynamics simulations indicate that conformational heterogeneity persists in ligand-bound complexes, the slow-timescale thermodynamic and kinetic landscape of these complexes in solution remains incompletely defined. Using 19F NMR spectroscopy, we characterize a slow-exchange conformational equilibrium in both the covalent nirmatrelvir (NMV) and noncovalent ensitrelvir (ENS) complexes of 3CLpro. For the wild-type 3CLpro–NMV complex at 298 K, joint dual-field line shape and exchange spectroscopy analysis resolves a millisecond-timescale exchange between two distinct states (population ratio ∼ 65:35; kex ≈ 52 s−1; ΔG‡ ≈ 15 kcal mol−1). Co-existing bound states also persist in both the noncovalent NMV–[C145A] mutant (minor state population, pB ≈ 18%) and wild-type ENS (pB ≈ 38%) complexes, demonstrating that this slow-exchange heterogeneity is an intrinsic property of the ligated protease rather than a consequence of covalent attachment. These dynamics are not readily explained by available crystal structures or conventional microsecond molecular dynamics simulations, suggesting that in solution the inhibited enzyme samples alternative, energetically accessible conformations that are not fully represented in the crystal lattice. This study highlights the utility of solution-state 19F NMR for quantifying low-energy conformational states relevant to drug–target energetics and inhibitor design.
For the integration of two-dimensional materials in future devices, a fundamental understanding of their response to external stimuli is needed. Toward this goal, we have investigated the electron and spin dynamics in the metallic van der Waals material Fe3GeTe2 (FGT) in its paramagnetic state after ultrafast optical excitation. To this end, we have employed a zone plate streaking technique with probing energies in the extreme ultraviolet range, tuned to the Fe M2,3 and Te N4,5 absorption edges. This approach provides insights into energy-dependent charge dynamics with a sensitivity to transient absorption changes on the order of ∼ 10 - 4 . We find a slow carrier relaxation time at both elemental edges-up to ( 2.2 ± 0.6 ) ps in Te and exceeding several picoseconds in Fe-which is surprising for a metal. To elucidate the complex time-resolved response, we also employ static x-ray absorption spectroscopy at the corresponding elemental edges, in which we find a double feature at the Fe M2,3 edge. We attribute this to different Fe sites in the pristine material and an oxidized surface layer, and we propose that the time-resolved absorption dynamics show a mixture of signals stemming from the different species. Additionally, we conducted time-resolved x-ray magnetic circular dichroism measurements in FGT at room temperature. We do not find clear evidence of the previously observed light-induced ferromagnetic order above T C . Our study lays the groundwork for a deeper understanding of charge and spin dynamics in FGT after optical excitation as part of a roadmap for 2D spintronics.
