Abstract
Clostridium perfringens forms metabolically dormant endospores that withstand extreme environmental conditions. Small acid-soluble proteins (SASPs) are ubiquitous DNA-binding proteins in endospores that promote resistance. While their protective role has been previously characterized, we aimed to provide further biophysical insight into the nature of these interactions, focusing on variant-specific structural dynamics through novel single-molecule and NMR approaches. Here, we characterize the DNA-binding properties and structural features of two Ssp4 variants using single-molecule fluorescence imaging and NMR spectroscopy along with electrophoretic mobility shift assays (EMSA). Both Ssp4 variants bind DNA cooperatively, but single-molecule analysis revealed preferential binding to GC-rich regions and significantly increased residence time in the presence of dipicolinic acid (DPA). NMR analysis reveals that an aspartic acid residue at position 36 (D36) stabilizes the Ssp4 structure, and its removal induces local structural perturbations without altering DNA affinity. Our findings provide molecular insights into how Ssp4 variants protect DNA in substantially dehydrated endospores and promote spore survival.