Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS.
Direct mass spectrometric characterization of interactions between proteins and small hydrophobic ligands often poses a serious problem due to the complex instability in the gas phase. We have developed a method that probes the efficacy of ligand-protein interactions indirectly by monitoring changes in protein flexibility. The latter is assessed quantitatively using a combination of charge state distribution analysis and amide hydrogen exchange under both native and mildly denaturing conditions. The method was used to evaluate binding of a model protein cellular retinoic acid binding protein I to its natural ligand all-trans retinoic acid (RA), isomers 13-cis- and 9-cis-RA, and retinol, yielding the following order of ligand affinities: All-trans RA > 9-cis RA > 13-cis RA, with no detectable binding of retinol. This order is in agreement with the results of earlier fluorimetric titration studies. Furthermore, binding energy of the protein to each of retinoic acid isomers was determined based on the measured hydrogen exchange kinetics data acquired under native conditions.
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Hui
H
Xiao
Igor A
IA
Kaltashov
Stephen J
SJ
Eyles
D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007474:Ion Exchange; D007536:Isomerism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D018168:Receptors, Retinoic Acid / Q000737:chemistry* / Q000378:metabolism*; D012176:Retinoids / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics
D001059:Apoproteins; D008024:Ligands; D018168:Receptors, Retinoic Acid; D012176:Retinoids; C082927:retinoic acid binding protein I, cellular; D003903:Deuterium