The kinetics of solvent accessibility at the protein-protein interface betw
een thrombin and a fragment of thrombomodulin, TMEGF45, have been monitored
by amide hydrogen/deuterium (H/H-2) exchange detected by MALDI-TOF mass sp
ectrometry. The interaction is rapid and reversible, requiring development
of theory and experimental methods to distinguish H/H-2 exchange due to sol
vent accessibility at the interface from H/H-2 exchange due to complex diss
ociation. Association and dissociation rate constants were measured by surf
ace plasmon resonance and amide H/H-2 exchange rates were measured at diffe
rent pH values and concentrations of TMEGF45. When essentially 100 % of the
thrombin was bound to TMEGF45, two segments of thrombin became completely
solvent-inaccessible, as evidenced by the pH insensitivity of the amide H/H
-2 exchange rates. These segments form part of anion-binding exosite I and
contain the residues for which alanine substitution abolishes TM binding. S
everal other regions of thrombin showed slowing of amide exchange upon TMEG
F45 binding, but the exchange remained pH-dependent, suggesting that these
regions of thrombin were rendered only partially solvent-inaccessible by TM
EGF45 binding. These partially inaccessible regions of thrombin form both s
urface and buried contacts into the active site of thrombin and contain res
idues implicated in allosteric changes in thrombin upon TM binding. (C) 200
1 Academic Press.