Jg. Mandell et al., Identification of protein-protein interfaces by decreased amide proton solvent accessibility, P NAS US, 95(25), 1998, pp. 14705-14710
Citations number
24
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Matrix-assisted laser desorption ionization-time-of-flight mass spectrometr
y was used to identify peptic fragments from protein complexes that retaine
d deuterium under hydrogen exchange conditions due to decreased solvent acc
essibility at the interface of the complex. Short deuteration times allowed
preferential labeling of rapidly exchanging surface amides so that primari
ly solvent accessibility changes and not conformational changes were detect
ed. A single mass spectrum of the peptic digest mixture was analyzed to det
ermine the deuterium content of all proteolytic fragments of the protein. T
he protein-protein interface was reliably indicated by those peptides that
retained more deuterons in the complex compared with control experiments in
which only one protein was present. The method was used to identify the ki
nase inhibitor [PKI(5-24)] and ATP-binding sites in the cyclic-AMP-dependen
t protein kinase. Three overlapping peptides identified the ATP-binding sit
e, three overlapping peptides identified the glycine-rich loop, and two pep
tides identified the PKI(5-24)-binding site. A complex of unknown structure
also was analyzed, human a-thrombin bound to an 83-aa fragment of human th
rombomodulin [TMEGF(4-5)]. Five peptides from thrombin showed significantly
decreased solvent accessibility in the complex. Three peptides identified
the anion-binding exosite I, confirming ligand competition experiments. Two
peptides identified a new region of thrombin near the active site providin
g a potential mechanism of how thrombomodulin alters thrombin substrate spe
cificity.