Free energies of protein-protein association determined by electrospray ionization mass spectrometry correlate accurately with values obtained by solution methods.
The advantages of electrospray ionization mass spectrometry (ESIMS) to measure relative solution-phase affinities of tightly bound protein-protein complexes are demonstrated with selected variants of the Bacillus amyloliquefaciens protein barstar (b*) and the RNAase barnase (bn), which form protein-protein complexes with a range of picomolar to nanomolar dissociation constants. A novel chemical annealing procedure rapidly establishes equilibrium in solutions containing competing b* variants with limiting bn. The relative ion abundances of the complexes and those of the competing unbound monomers are shown to reflect the relative solution-phase concentrations of those respective species. No measurable dissociation of the complexes occurs either during ESI or mass detection, nor is there any evidence for nonspecific binding at protein concentrations < 25 microM. Differences in DeltaDeltaG of dissociation between variants were determined with precisions < 0.1 kcal/mol. The DeltaDeltaG values obtained deviate on average by 0.26 kcal/mol from those measured with a solution-phase enzyme assay. It is demonstrated that information about the protein conformation and covalent modifications can be obtained from differences in mass and charge state distributions. This method serves as a rapid and precise means to interrogate protein-protein-binding surfaces for complexes that have affinities in the picomolar to nanomolar range.
Krishnaswamy
Sanjay R
SR
Program in Biophysics, University of California at Berkeley, Berkeley, California 94720-1460, USA.
Williams
Evan R
ER
Kirsch
Jack F
JF
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D007477:Ions; D010084:Oxidation-Reduction; D025941:Protein Interaction Mapping / Q000379:methods*; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014508:Urea / Q000737:chemistry
D001426:Bacterial Proteins; D007477:Ions; D012996:Solutions; C057438:barstar protein, Bacillus amyloliquefaciens; D014508:Urea; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease