ABSTRACT
Hydrogen-deuterium exchange (HDx) associated with mass spectrometry (MS) is emerging as a powerful tool to provide conformational information about membrane proteins. Unfortunately, as for X-ray diffraction and NMR, HDx performed on reconstituted in vitro systems might not always reflect the in vivo environment. Outer-membrane vesicles naturally released by Escherichia coli were used to carry out analysis of native OmpF through HDx-MS. A new protocol compatible with HDx analysis that avoids hindrance from the lipid contents was setup. The extent of deuterium incorporation was in good agreement with the X-ray diffraction data of OmpF as the buried ß-barrels incorporated a low amount of deuterium, whereas the internal loop L3 and the external loops incorporated a higher amount of deuterium. Moreover, the kinetics of incorporation clearly highlights that peptides segregate well in two distinct groups based exclusively on a trimeric organization of OmpF in the membrane: peptides presenting fast kinetics of labeling are facing the complex surrounding environment, whereas those presenting slow kinetics are located in the buried core of the trimer. The data show that HDx-MS applied to a complex biological system is able to reveal solvent accessibility and spatial arrangement of an integral outer-membrane protein complex.
