DIRECT MEASUREMENT OF OLIGONUCLEOTIDE BINDING STOICHIOMETRY OF GENE-VPROTEIN BY MASS-SPECTROMETRY

The binding stoichiometry of gene V protein from bacteriophage f1 to s everal oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditi ons that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide comp lexes with stoichiometry of approximately four nucleotides (nt) per pr otein monomer. A 16-mer oligonucleotide gave predominantly: a 1:1 (pro tein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily meas ured using mass spectrometry; The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size -exclusion chromatography and the results were consistent with the mas s spectrometric data. These results demonstrate, for the first time, t he observation and stoichiometric measurement of protein-oligonucleoti de complexes using ESI-MS. The sensitivity and high resolution of ESI- MS should make it a useful tool in the study of protein-DNA interactio ns.