Response Factor Correction for Quantitative Determination of Homooligomeric SsoSSB Binding to ssDNA by Native Mass...

Native mass spectrometry (nMS) has emerged as a complementary approach for elucidating molecular parameters of biological complexes relative to solution-phase experiments. Herein, we utilize nMS to determine the subunit binding affinities (K_d,i) of the single-stranded DNA binding protein (SSB) from Saccharolobus solfataricus (Sso) to poly dT single-stranded DNA (ssDNA) compared with the apparent K_d' values obtained from solution-phase fluorescence anisotropy. This work resolves conflicting previous biochemical reports on the stoichiometry and affinities of SsoSSB while also highlighting the advantages and limitations of nMS quantification. Covalent concatemers of SsoSSB with increasing molecular weights were utilized as response factor (RF) standards to correct for physical and instrumental parameters that systematically underrepresent abundances from ionization of larger mass species. Furthermore, we show that regardless of the nMS quantification metric (peak area or intensity), meaningful comparative data can be extracted from multicomponent biochemical systems. Importantly, the binding affinities of the individual species (K_d,i) determined by nMS approach the apparent K_d' from bulk solution-phase measurements but have the added benefit of separately quantifying individual binding steps within a multistep assembly process. Interestingly, the stoichiometries of SsoSSB binding to 15 or 30 nucleotides of ssDNA measured by nMS are subsaturating. The calculated binding affinities of the first and second SsoSSB molecules show some positive cooperativity, while the binding of the third is an order of magnitude weaker, suggesting that negative cooperativity is utilized to limit binding near the ends of the available length of the ssDNA.