Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

Abstract

The binding stoichiometry of gene V protein from bacteriophage ft 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 proteinoligonucleotide 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 proteinoligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful tool in the study of protein-DNA interactions.Protein-DNA interactions are involved in many cellular processes and thus are fundamentally important for the functioning of biological systems. The stoichiometry of binding is an important characteristic of protein-DNA interactions. Determination of binding stoichiometries can be difficult when there are mixed stoichiometries present in a sample or when molecular mass (Mr) differences between possible alternative stoichiometries are small (1). Mass spectrometry (MS) is the most accurate method for Mr measurement, and thus is potentially useful for accurate determination of protein-DNA binding stoichiometry. However, the application of MS to the direct characterization of protein-DNA complexes has been precluded due to the difficulty of transferring these complexes intact from solution into the mass spectrometer using conventional ionization methods. Electrospray ionization (ESI) allows ionization of non-volatile samples from solution and the development of this technique has greatly expanded the application of mass spectrometry in the analysis of biomolecules (2-5). Recently, a number of groups have reported observations of specific noncovalent complexes of biomolecules from solution using ESI-MS (6-11). These reports prompted us to investigate the application of this technique to intact protein-DNA noncovalent complexes.We chose gene V protein from bacteriophage fl for our study due to its well characterized structure (12-16) and the extensive studies on the DNA binding stoichiometry and cooperatively (17)(18)(19)(20)(21)(22). The gene V protein stabilizes singlestranded DNA (ssDNA) during phage replication, and shifts the synthesis of phage DNA from double-stranded form to single-stranded form, in the. later stage of phage infection, by preventing the synthesis of th...

Characterization of noncovalent protein-ligand complexes and associated enzyme intermediates of GlcNAc-6-O-sulfotransferase by electrospray ionization FT-ICR mass spectrometry

“…The mass differences between these two new ions and NodST were calculated to be 507 Da and 427 Da, which correspond to the noncolvalent adduction of PAPS and PAP, respectively. The five-fold excess of ligands was mixed with the protein so that both the binding and its specificity can be assessed [27]. Only one molecule each of the substrate (PAPS) and the inhibitor (PAP) was observed to be bound to the protein, even under the conditions of molar excess of the ligands.…”

Evaluation of protein-DNA binding affinity by electrospray ionization mass spectrometry

“…Compared with protein-ligand or multimeric protein complexes, fewer studies were reported on protein-DNA noncovalent complexes by ESI-MS [13][14][15][16][17][18], although protein-DNA interactions play essential roles in a variety of significant biological processes, such as gene-expression regulation, DNA repair and replication, and transcription regulation, etc. In the protein-DNA interactions, electrostatic forces and hydrogen bonds significantly contribute to the binding of the complex in addition to hydrophobic interactions.…”

A eole for the MS analysis of nucleic acids in the post-genomics age

“…Direct ESI-MS analysis of nucleic acid complexes with other nucleic acids [106 -110], proteins [111][112][113][114][115][116], and small molecule ligands [117][118][119][120] can reveal their exact composition and stoichiometry from the observed molecular mass, dispensing with the typical curvefitting of bulk data required by spectroscopic and calorimetric methods. Unlike these techniques, MS is capable of resolving any free/bound species at equilibrium in solution, even when such species possess very similar spectroscopic characteristics.…”