Stoichiometries of protein-protein/DNA binding and conformational changes for the transition-state regulator AbrB measured by pseudo cell-size exclusion chromatography-mass spectrometry.
We have developed on-line pseudo cell-size exclusion chromatography-mass spectrometry (PsC-SEC-MS) for the rapid, real time analyses of noncovalently bound protein complexes. The methodology can be used to determine constituent components of such complexes, as well as exact stoichiometries. Furthermore, it enables the efficient determination of gross conformational changes upon complexation. The power of the new approach is demonstrated in the analysis of the global transition-state regulator AbrB and its complex with a target DNA sequence from the promoter sinIR. Using PsC-SEC-MS, we confirm that AbrB is assembled as a homotetramer and not as a homohexamer as previously suggested. Additionally, we show that AbrB binds to the sinIR DNA target element as a homotetramer, affording a 4:1 protein:DNA stoichiometry. Finally, we demonstrate that when the complex binds to sinIR, the hydrodynamic volume (size) of the complex is notably reduced compared to that of the apoprotein, indicating a protein conformational change.
Cavanagh
John
J
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA. john_cavanagh@ncsu.edu
Thompson
Richele
R
Bobay
Benjamin
B
Benson
Linda M
LM
Naylor
Stephen
S
D001412:Bacillus subtilis / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D002850:Chromatography, Gel / Q000295:instrumentation / Q000379:methods; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004579:Electron Transport; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins / Q000737:chemistry; D001411:Geobacillus stearothermophilus / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods; D014157:Transcription Factors / Q000737:chemistry*
C060762:AbrB protein, Bacillus subtilis; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; C097712:SinI protein, Bacillus subtilis; D014157:Transcription Factors; C076733:FlaD protein, Bacteria; D004247:DNA