Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.
The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions 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 complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein 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 measured 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 mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.
Cheng
X
X
Environmental Molecular Sciences laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Harms
A C
AC
Goudreau
P N
PN
Terwilliger
T C
TC
Smith
R D
RD
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism*; D011061:Poly A; D011071:Poly T; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; C069558:gene V protein, Enterobacteria phage f1; D011061:Poly A; D011071:Poly T