Bin Xia

Protein functions are closely linked to their dynamic behaviors, particularly involving low-abundance transient states. Quantifying these interconversions remains experimentally challenging, especially for proteins containing intrinsically disordered regions (IDRs). In this study, we utilize low-dose electron microscopy using graphene liquid cells to image and quantify interconversions between protein conformational states under optimized electron beam conditions and confinement effects. Applied to four proteins in solution: CRISPR-associated protein (Cas9), DNA binding protein (GapR), restriction enzyme (EcoRI), and an RNA binding protein with IDRs (FUS), we visualized low-abundance transient structures distinct from crystalline forms and elucidated transition pathways which are validated by cryo-electron microscopy, revealing differences in their energy landscapes through single-molecule imaging. We analyzed domain-scale rearrangements at ∼1–2 nm effective conformational resolution, cross-validated against independent computational and experimental methods. Shifts in state equilibria and conformational adaptations were observed when proteins are mutated or during biomolecular interactions, such as target searching, binding, and cleavage with nucleic acids. Notably, changes in the energy landscape were observed when proteins with IDRs and RNA were within close proximity but not in apparent contact, suggesting that conformational redistribution may precede complete binding. This study shows how to characterize protein conformations and their time-dependent changes using instrumentation that is widely available at research universities but not yet used for these purposes.

The locus of enterocyte effacement-encoded regulator (Ler) is a master transcriptional activator essential for the virulence of enterohemorrhagic and enteropathogenic Escherichia coli. Although Ler shares homology with the global silencer H-NS, it functions uniquely as an anti-silencer, a role strictly dependent on its oligomerization state. However, the structural mechanism governing Ler assembly remains poorly understood. In this study, we have characterized the N-terminal oligomerization domain (Ler1–74) of Ler using solution NMR spectroscopy and biophysical assays, and found that Ler1–74 shows concentration-dependent oligomerization. We demonstrate that Ler oligomerization is driven by two distinct interfaces with contrasting dynamic properties. We determined the solution structure of the Ler18–74 dimer, revealing a stable, anti-parallel “tail-to-tail” interface (dimer Site-2, residues 35–66) stabilized by a hydrophobic core. In contrast, the N-terminal interface (dimer Site-1, residues 12–33) forms a highly dynamic “head-to-head” dimer, which undergoes significant conformational exchange and exhibits concentration- and temperature-dependent dimerization. Based on these findings, we propose a structural model wherein Ler forms supramolecular assemblies through the propagation of alternating stable (Site-2) and dynamic (Site-1) interactions. This architecture, while reminiscent of H-NS, displays distinct stability features that may underlie Ler's specific anti-silencing function in bacterial pathogenesis.

Fan Liu, Jing Xu, Xinli Hu, Bo Duan, Bin Xia; Quantitative real-time in vitro transcription assay (QRIVTA) for transcriptional regulation studies, Protein

SarA is a global transcription regulator in S. aureus which regulates the expression of over 120 genes related to quorum sensing, biofilm synthesis, drug resistance and many other important physiological processes during host infection. SarA can bind to the promoter region of agr and other target genes to activate or repress the transcription. The crystal structure of SarA uncovered a MarR protein-like conformation with two symmetrical winged helix domains, while its DNA binding mechanism is still unknown. We have constructed a monomeric DNA binding domain of SarA (SarA^ΔN19) for the study of the interaction between SarA and DNA with NMR spectroscopy. Here, we report the ¹H, ¹³C and ¹⁵N NMR assignment of SarA^ΔN19/DNA complex which is the first step towards further structure and function analysis.