This perspective highlights recent applications and technological progress in dipolar electron paramagnetic resonance (EPR) spectroscopy, including double electron-electron resonance (DEER) spectroscopy. These methods provide nanoscale distance distributions between site-specific spin labels in biomacromolecules. The resulting data are particularly well suited for quantifying the structure and energetics of conformational ensembles of multi-state and flexible proteins. Recent applications span a wide range of systems and are accompanied by innovations in spin labeling, deuteration, in-cell measurements, integrative multi-technique approaches, and novel computational modeling methods combined with structure prediction tools.

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate, a bioactive lipid implicated in cancer progression and other diseases. Despite its clinical relevance, the structural and dynamic basis of SK1 regulation and inhibition remains poorly understood. Using an integrated spectroscopic and computational approach, we uncover conformational transitions that govern substrate entry, catalysis, and inhibitor binding. Phosphorylation of Ser225 triggers regulatory loop rearrangements and salt bridge reshuffling, priming SK1 for membrane engagement and catalytic activity. We identify a previously uncharacterized catalytic intermediate featuring a distinct conformation with a highly dynamic lipid-binding loop 1 (LBL-1), sensitive to potent inhibitors such as PF-543. This inhibitor locks SK1 in an inactive state by restricting LBL-1 dynamics and globally stabilizing a non-catalytic conformation. Notably, SK1 forms functionally distinct dimers stabilized by ligand or membrane interactions, revealing a dynamic, multilayered regulatory mechanism governed by structural flexibility. These findings define a novel inhibitory mechanism and offer a structural framework for developing next-generation SK1-targeted therapeutics.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is now a critical tool in molecular biology and structural proteomics. It is routinely used to probe protein and conformational dynamics through a well-established experiment where amide hydrogens exchange with deuterium atoms in a buffer containing DO. Although there have been numerous advances in the field, data analysis still poses challenges mainly due to the need for manual curation of the data and the lack of standardized statistics and accessible software. In response, we developed Kingfisher, an open-source, user-friendly, web-based solution that facilitates downstream analysis using well-established statistics and provides advanced high-resolution representations of the HDX results. Kingfisher is able to read data directly as exported from common software packages and usually takes less than a minute to run the analysis, without the need to download the raw code or install any software. We foresee Kingfisher as a valuable tool for both newcomers and experts in the field of Hydrogen Exchange Mass Spectrometry. Kingfisher is available to all users as an interactive web application at https://kingfisher.wustl.edu/.

The partner and localizer of BRCA2 (PALB2) is a scaffold protein linking BRCA1 with BRCA2 and RAD51 during homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR in cells, while the PALB2 DNA-binding domain (PALB2-DBD) supports DNA strand exchange . We determined that PALB2-DBD is intrinsically disordered beyond a single N-terminal α-helix. Coiled-coil mediated dimerization is stabilized by interaction between intrinsically disordered regions (IDRs) leading to a 2-fold structural compaction. Single-stranded (ss)DNA binding promotes additional structural compaction and protein tetramerization. Using confocal single-molecule FRET, we observed bimodal and oligomerization-dependent compaction of ssDNA bound to PALB2-DBD, suggesting a novel strand exchange mechanism. Bioinformatics analysis and preliminary observations indicate that PALB2 forms protein-nucleic acids condensates. Intrinsically disordered DBDs are prevalent in the human proteome. PALB2-DBD and similar IDRs may use a chaperone-like mechanism to aid formation and resolution of DNA and RNA multichain intermediates during DNA replication, repair and recombination.

Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and double electron-electron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.