Integral membrane proteins are embedded in the biological
membrane,
where they carry out numerous biological processes. Although lipids
present in the membrane are crucial for membrane protein function,
it remains difficult to characterize many lipid binding events to
membrane proteins, such as those that form the annular belt. Here,
we use native mass spectrometry along with the charge-reducing properties
of trimethylamine N-oxide (TMAO) to characterize
a large number of lipid binding events to the bacterial ammonia channel
(AmtB). In the absence of TMAO, significant peak overlap between neighboring
charge states is observed, resulting in erroneous abundances for different
molecular species. With the addition of TMAO, the weighted average
charge state (Zavg) was decreased. In
addition, the increased spacing between nearby charge states enabled
a higher number of lipid binding species to be observed while minimizing
mass spectral peak overlap. These conditions helped us to determine
the equilibrium binding constants (Kd)
for up to 16 lipid binding events. The binding constants for the first
few lipid binding events display the highest affinity, whereas the
binding constants for higher lipid binding events converge to a similar
value. These findings suggest a transition from nonannular to annular
lipid binding to AmtB