In situ Investigation of HdeA in Bacterial Outer Membrane Vesicles Using NMR Spectroscopy

Abstract

HdeA, a molecular chaperone localized in the bacterial periplasm, plays a vital role in maintaining protein homeostasis. Previous studies of HdeA were mainly carried out in in vitro conditions, limiting our understanding of the mechanism of HdeA in its native environment. Outer membrane vesicles of bacteria are extracellular vesicles released by cells into the extracellular milieu in a controlled manner, with contents almost identical to the periplasmic environment. Thus, by enriching HdeA into the bacterial outer membrane vesicles (OMVs), we investigated the conformational changes of HdeA within OMVs via nuclear magnetic resonance (NMR) spectroscopy. The results reveal that HdeA exhibits an acid-dependent conformational change in its native environment. The high-resolution spectrum of HdeA in OMVs indicates that under low pH conditions the function of HdeA is activated through residues S15, W16, T17, S27, T32, E36, G54, T57, C66, Q71, F74 and D83. Moreover, the NMR measurement of HdeA within OMVs provides a promising way for in situ investigation of mechanisms of other periplasmic molecular chaperones via NMR spectroscopy.

Keywords

• in situ nmr
• hdea
• outer membrane vesicles
• chaperone