title: MALDI-MS detection of noncovalent interactions of single stranded DNA with Escherichia coli single-stranded DNA-binding protein.
abstract: 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.
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zürich, CH-8093 Zürich, Switzerland.
authors.forename: Fan
authors.initials: F
authors.lastname: Chen
authors.forename: Stefanie
authors.initials: S
authors.lastname: Mädler
authors.forename: Simon
authors.initials: S
authors.lastname: Weidmann
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D055672:Static Electricity
chemical_list: D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C476430:SSB protein, E coli