2 research outputs found
Binding States of Protein–Metal Complexes in Cells
The
identification of endogenous proteins as well as their binding
to metal ions in living cells is determined by combining pulsed electrophoretic
separations with nanoelectrospray ionization followed by mass spectrometric
detection. This approach avoids problems resulting from the complicated
cellular environment. In this manner, we demonstrate the rapid identification
(300 ms or less) of intact proteins from living E.
coli cells including the complexation of calmodulin
with calcium ion. The latter showed different binding states from
those observed in in vitro studies. These observations also reveal
in vitro measurements do not necessarily represent the actual situation
in living cells. We conclude that the attempted in situ measurement
of intracellular proteins with minimal sampling processes should be
preferred
Reliable Tracking In-Solution Protein Unfolding via Ultrafast Thermal Unfolding/Ion Mobility-Mass Spectrometry
Sequential
unfolding of monomeric proteins is important for the
global understanding of local conformational elements (e.g., secondary
structures and domain connections) within those protein assemblies.
Ion mobility-mass spectrometry (IM-MS) is an emerging and promising
technique for probing gradual protein structural perturbations in
the gas phase. However, it is still challenging to track sequential
unfolding in the solution phase. Here, we extended IM-MS to track
in-solution sequential unfolding of monomeric proteins having single
and/or multidomains. The present method combines ultrafast local heating
effect (LHE)-driven sequential unfolding with IM-MS identification.
Protein sequential unfolding in solution is demonstrated by the rapid
and controllable IM-MS data switch between native and gradually unfolded
states. Our results show that LHE induces gradual protein conformational
transitions associated with biological functions, where IM-MS tracks
the sequential unfolding of monomeric proteins