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.
Polysaccharide structural assignment via nuclear magnetic resonance (NMR) spectroscopy remains an analytical challenge due to spectral overlap because of limited chemical shift dispersion. This challenge is exacerbated by the wide-spread use of proton (1H) detection. Progress has also been hindered by the dispersed nature of carbohydrate databases and the restricted applicability of most prediction tools, which provide limited atom-specific residue discrimination in larger polymers. These limitations hamper the efficient characterization of glycosyltransferase (GT) activity, which depends on defining donor-acceptor substrate pairs and accurately identifying corresponding products. Here, we demonstrate the utility of high field 13C-detected NMR for the assignment of two homohexamers. Laminarihexaose and xylohexaose were analyzed using 2D heteronuclear correlation and correlation via long-range coupling (COLOC) experiments to evaluate whether complete residue level assignments could be achieved. The COLOC experiment revealed long range correlations that were not resolved using heteronuclear multiple bond correlation, and the 1D 13C spectra provided exceptional resolution, including distinct shoulders corresponding to residue specific chemical shift differences previously assumed to be indistinguishable. These findings suggest that high field 13C NMR can provide the nuanced atom level information required to train machine learning models for predicting chemical shift assignments of large, complex, and highly degenerate polysaccharides. Such models offer a promising framework for rapid structural identification of GT reaction products, enabling progress of high throughput characterization of plant derived polysaccharides central to renewable biomaterial development.
Strain engineering in epitaxial Bi(111) films on Si(111) enables temperature-tunable phonon frequencies through thermal expansion mismatch. Using in situ low-energy electron diffraction combined with broadband femtosecond transient reflectivity on the same samples, we directly correlate anisotropic lattice parameters with the redshift of the coherent A1g phonon mode. The in-plane lattice parameter remains locked to the Si substrate and is therefore nearly temperature-independent, imposing strong biaxial compressive strain, causing an additional expansion of the Bi lattice along the c-axis. By applying an extended phonon-shift model, we disentangle the contributions of intrinsic thermal expansion, thermal-mismatch strain, and anharmonicity to the phonon softening. The enhanced redshift observed in strained films cannot be accounted for by expansion or mismatch alone; instead, it arises from a strengthened anharmonic term that additionally scales with strain along the c-axis, being insensitive to the in-plane lattice parameter and thereby enabling targeted control of phonon frequencies.
