Fan Chen1, Stefanie Mädler2, Simon Weidmann1, and Renato Zenobi1
- Department of Chemistry and Applied Biosciences, ETH Zürich, CH-8093 Zürich, Switzerland
- Centre for Research in Mass Spectrometry, York University, Toronto, ON M3J 2R7, Canada
The Escherichia coli single-stranded DNA binding protein (SSB) selectively binds single-stranded (ss) DNA and participates in the process of DNA replication, recombination and repair. Different binding modes have previously been observed in SSB•ssDNA complexes, due to the four potential binding sites of SSB. Here, chemical cross-linking, combined with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), is used to determine the stoichiometry of the SSB•ssDNA complex. SSB forms a stable homotetramer in solution, but only the monomeric species (m/z 19 100) can be detected with standard MALDI-MS. With chemical cross-linking, the quaternary structure of SSB is conserved, and the tetramer (m/z 79 500) was observed. We found that ssDNA also functions as a stabilizer to conserve the quaternary structure of SSB, as evidenced by the detection of a SSB•ssDNA complex at m/z 94 200 even in the absence of chemical cross-linking. The stability of the SSB•ssDNA complex with MALDI strongly depends on the length and strand of oligonucleotides and the stoichiometry of the SSB•ssDNA complex, which could be attributed to electrostatic interactions that are enhanced in the gas phase. The key factor affecting the stoichiometry of the SSB•ssDNA complex is how ssDNA binds to SSB, rather than the protein-to-DNA ratio. This further suggests that detection of the complex by MALDI is a result of specific binding, and not due to non-specific aggregation in the MALDI plume.
CovalX Technology Used (Click each option to learn more)
10 μL of a 2 μM protein solution (monomer concentration) was incubated with 1 μL of DSS dissolved in DMF for 1 hour at room temperature in order to chemically cross-link SSB from E. coli. 1 μ: of DMF was added to another 10 μL SSB solution to use as a control. 10 μL of the 2 μM protein solution was mixed with 1 μL of 5 μM poly-d(T)n at room temperature for 15 minutes and then incubated for 1 hour at room temperature with a DSS solution (10/1, v/v, protein/DSS). A variety of poly-d(T)n concentrations were mixed with the same protein solution in a volume of 1/10 (v/v, DNA/protein) and a control experiment without a cross-linker was created.
SA was dissolved at 5 mg/mL and 20 mg/mL in water/acetonitrile/TFA (49.95/49.95/0.1, v/v/v) to create the matrix. The 5 mg/mL solution was spotted on a stainless steel plate and dried in ambient conditions before 0.5 μL of sample solution without matrix was added. Finally, a second layer of the SA solution at 20 mg/mL was added on top of the sample solution layer after it had dried. The plates were then analyzed by MALDI-TOF/TOF mass spectrometer that had been modified with a CovalX HM2 detection system. From this analysis, the researchers were able to understand the stoichiometry of SSB-ssDNA complexes and observe protein-DNA complexes for the first time. They were also able to determine the stability and estimate the accessible number of amine groups.