Determination of Protein-Ligand Binding Constants of a Cooperatively Regulated Tetrameric Enzyme Using Electrospray Mass Spectrometry

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Author(s) Cubrilovic, Dragana, Haap, Wolfgang, Barylyuk, Konstantin, Ruf, Armin, Badertscher, Martin, Gubler, Marcel, Tetaz, Tim, Joseph, Catherine, Benz, Jörg, Zenobi, Renato
Publication Type Journal Items, Publication Status: Published
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Title Determination of Protein-Ligand Binding Constants of a Cooperatively Regulated Tetrameric Enzyme Using Electrospray Mass Spectrometry
Author(s) Cubrilovic, Dragana
Haap, Wolfgang
Barylyuk, Konstantin
Ruf, Armin
Badertscher, Martin
Gubler, Marcel
Tetaz, Tim
Joseph, Catherine
Benz, Jörg
Zenobi, Renato
Journal or Series Title ACS chemical biology
Volume Number 9
Issue Number 1
Start Page 218
End Page 226
ISSN 1554-8929
1554-8937
Publisher American Chemical Society
Publication Place Columbus, OH
Publication Date 2014
Abstract This study highlights the benefits of nano electrospray ionization mass spectrometry (nanoESI-MS) as a fast and label-free method not only for determination of dissociation constants (K-D) of a cooperatively regulated enzyme but also to better understand the mechanism of enzymatic cooperativity of multimeric proteins. We present an approach to investigate the homotetrameric enzyme fructose 1,6-bisphosphatase (FBPase), a allosteric mechanism in the binding of inhibitors to the potential therapeutic target for glucose control in type 2 diabetes. A series of inhibitors binding at an allosteric site of FBPase were investigated to determine their K(D)s by nanoESI-MS. The K(D)s determined by ESI-MS correlate very well with IC50 values in solution. The Hill coefficients derived from nanoESI-MS suggest positive cooperativity. From single-point measurements we could obtain information on relative potency, stoichiometry, conformational changes, and mechanism of cooperativity. A new X-ray crystal structure of FBPase tetramer binding ligand 3 in a 4:4 stoichiometry is also reported. NanoESI-MS-based results match the current understanding of the investigated system and are in agreement with the X-ray structural data, but provide additional mechanistic insight on the ligand binding, due to the better dynamic resolution. This method offers a powerful approach for studying other proteins with allosteric binding sites, as well.
DOI 10.1021/cb4007002
Document Type Article
Publication Status Published
Language English
Assigned Organisational Unit(s) 03430
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NEBIS System Number 005076119
Source Database ID WOS-000267297700007
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@article{Cbrlvc2014,
  author = "Cubrilovic, Dragana and Haap, Wolfgang and Barylyuk, Konstantin and Ruf, Armin and Badertscher, Martin and Gubler, Marcel and Tetaz, Tim and Joseph, Catherine and Benz, J{\"{o}}rg and Zenobi, Renato",
  title = "{D}etermination of {P}rotein-{L}igand {B}inding {C}onstants of a {C}ooperatively {R}egulated {T}etrameric {E}nzyme {U}sing {E}lectrospray {M}ass {S}pectrometry",
  journal = "ACS chemical biology",
  year = 2014,
  volume = "9",
  number = "1",
  pages = "218--226",
}


E-Citations record created: Mon, 03 Mar 2014, 13:54:58 CET