Intact noncovalent dimer of estrogen receptor ligand-binding domain can be detected by electrospray ionization mass spectrometry.
Electrospray ionization mass spectrometry (ESMS) of the estrogen receptor ligand binding domain (ER LBD) in its estradiol-binding form was performed. A dimeric ER LBD was observed, with a greatly reduced capacity for protonation (major charge state for dimer +16 vs. +23 for a monomer). Peak broadening (probably due to heterogeneity resulting from salt and water adduct formation) adversely affected our ability to distinguish between multiple discreet dimeric species and thus prevented us from establishing an accurate average mass for the dimerized domain. A mixture of species with molecular masses between 57,240 Da and 57,900 Da was observed, which would compare to 57,274 Da, 57,546 Da, and 57,818 Da for the calculated masses of the dimer without estradiol, or with one or two bound ligand molecules, respectively. Hence, nonliganded ER LBD dimer appeared to constitute the major species. The presence of low levels of a singly liganded ER LBD dimer cannot be ruled out, but the data argue against the possibility of the ER LBD dimer carrying two molecules of estradiol. Allowing for current limitations in the technology, our data demonstrate that ESMS on a quadrupole mass spectrometer of limited mass range (4000 Da for singly charged ions) has potential utility for studying ligand-binding proteins. In particular, in future it might be possible to compare spectra obtained from agonist- and antagonist-bound receptors and determine from subtle changes in protonation state possible differences in the higher order structure of those noncovalent protein complexes.
Witkowska
H E
HE
Children's Hospital Oakland Research Institute, California 94609, USA.
Green
B N
BN
Carlquist
M
M
Shackleton
C H
CH
D001665:Binding Sites; D004958:Estradiol / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000378:metabolism*; D012997:Solvents
D008024:Ligands; D011960:Receptors, Estrogen; D012997:Solvents; D004958:Estradiol