Speaker
Description
Single-stranded DNA-binding proteins (SSBs) are essential for genome maintenance across all domains of life, protecting transiently exposed single-stranded DNA during replication, recombination, and repair. While the crystal structure and biophysical properties of SsbA from Streptomyces coelicolor have been characterized, direct information on its solution-phase conformational dynamics has been lacking.
In this study, hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to probe the conformational dynamics of free SsbA in physiological conditions. Deuterium labeling was performed at five time points (10 s, 1 min, 20 min, 1 h, 4 h). Two proteolytic strategies were used to generate overlapping peptide maps, and data were analyzed using HDExaminer software, yielding approximately 70% sequence coverage. Regions corresponding to the N-terminal His-tag (residues 1–9) and portions of the C-terminal region (residues 152–211), which includes glycine rich region and acidic C-tip (DEPPF) were not covered, consistent with known challenges in peptide identification within low-complexity sequences
The N-terminal OB-fold domain, responsible for ssDNA binding, showed a heterogeneous exchange pattern, with the highest deuterium uptake observed in the region spanning residues 26–44, and additional regions of elevated exchange around residues 60–66 and 72–89, revealing locally flexible or solvent-exposed segments. In contrast, the glycine-rich IDL region (residues ~121–151), previously described as intrinsically disordered, exhibited consistently low deuterium uptake across all time points, suggesting reduced solvent accessibility in the free state relative to that expected for a fully disordered linker. This observation is consistent with previously reported hydrodynamic data suggesting a more globular conformation of the SsbA C-domain, and may reflect self-association or condensate-forming behavior proposed for this protein.
These findings provide the first direct evidence of the conformational dynamics of free SsbA in solution and establish a baseline for future comparative HDX-MS studies.
Keywords: SsbA, HDX-MS, Streptomyces coelicolor, conformational dynamics