Giuseppe Licari, Karolina Strakova, Stefan Matile, and Emad Tajkhorshid.
Twisting and tilting of a mechanosensitive molecular probe detects
order in membranes.
Chemical Science, 11:5637-5649, 2020.
(PMC: PMC7433777)
LICA2020-ET
Lateral forces in biological membranes affect a variety of dynamic cellular processes. Recent
synthetic efforts have introduced fluorescent "flippers'' as environment-sensitive
planarizable push-pull probes that can detect lipid packing and membrane tension, and
respond to lipid-induced mechanical forces by a shift in their spectroscopic properties.
Herein, we investigate the molecular origin of the mechanosensitivity of the best known
flipper, Flipper-TR, by an extended set of molecular dynamics (MD) simulations in membranes
of increasing complexity and under different physicochemical conditions, revealing
unprecedented details of the sensing process. Simulations enabled by accurate refinement of
Flipper-TR force field using quantum mechanical calculations allowed us to unambiguously
correlate the planarization of the two fluorescent flippers to spectroscopic response. In
particular, Flipper-TR conformation exhibits bimodal distribution in disordered membranes
and a unimodal distribution in highly ordered membranes. Such dramatic change was
associated with a shift in Flipper-TR excitation spectra, as supported both by our simulated
and experimentally-measured spectra. Flipper-TR sensitivity to phase-transition is confirmed
by a temperature-jump protocol that alters the lipid phase of an ordered membrane,
triggering an instantaneous mechanical twisting of the probe. Simulations show that the
probe is also sensitive to surface tension, since even in a naturally disordered membrane, the
unimodal distribution of coplanar flippers can be achieved if a sufficiently negative surface
tension is applied to the membrane. MD simulations in ternary mixtures containing raft-like
nanodomains show that the probe can discriminate lipid domains in phase-separated complex
bilayers. A histogram-based approach, called DOB-phase classification, is introduced that
can differentiate regions of disordered and ordered lipid phases by comparing dihedral
distributions of Flipper-TR. Moreover, a new sensing mechanism involving the orientation of
Flipper-TR is elucidated, corroborating experimental evidence that the probe tilt angle is
strongly dependent on lipid ordering. The obtained atomic-resolution description of Flipper-
TR mechanosensitivity is key to the interpretation of experimental data and to the design of
novel mechanosensors with improved spectroscopic properties.
