Self-assembled proteoliposomes allow highly efficient energy transfer from the spectrally-complementary chromophore Texas Red to the plant light-harvesting protein LHCII, increasing the effective absorption range of this bio-hybrid system.
Natural photosynthetic “thylakoid” membranes found in green plants contain a large network of light‐harvesting (LH) protein complexes. Rearrangement of this photosynthetic machinery, laterally within stacked membranes called “grana”, alters protein–protein interactions leading to changes in the energy balance within the system. Preparation of an experimentally accessible model system that allows the detailed investigation of these complex interactions can be achieved by interfacing thylakoid membranes and synthetic lipids into a template comprised of polymerized lipids in a 2D microarray pattern on glass surfaces. This paper uses this system to interrogate the behavior of LH proteins at the micro‐ and nanoscale and assesses the efficacy of this model. A combination of fluorescence lifetime imaging and atomic force microscopy reveals the differences in photophysical state and lateral organization between native thylakoid and hybrid membranes, the mechanism of LH protein incorporation into the developing hybrid membranes, and the nanoscale structure of the system. The resulting model system within each corral is a high‐quality supported lipid bilayer that incorporates laterally mobile LH proteins. Photosynthetic activity is assessed in the hybrid membranes versus proteoliposomes, revealing that commonly used photochemical assays to test the electron transfer activity of photosystem II may actually produce false‐positive results.
Fluorescent probes
are useful in biophysics research to assess
the spatial distribution, mobility, and interactions of biomolecules.
However, fluorophores can undergo “self-quenching” of
their fluorescence intensity at high concentrations. A greater understanding
of concentration-quenching effects is important for avoiding artifacts
in fluorescence images and relevant to energy transfer processes in
photosynthesis. Here, we show that an electrophoresis technique can
be used to control the migration of charged fluorophores associated
with supported lipid bilayers (SLBs) and that quenching effects can
be quantified with fluorescence lifetime imaging microscopy (FLIM).
Confined SLBs containing controlled quantities of lipid-linked Texas
Red (TR) fluorophores were generated within 100 × 100 μm
corral regions on glass substrates. Application of an electric field
in-plane with the lipid bilayer induced the migration of negatively
charged TR-lipid molecules toward the positive electrode and created
a lateral concentration gradient across each corral. The self-quenching
of TR was directly observed in FLIM images as a correlation of high
concentrations of fluorophores to reductions in their fluorescence
lifetime. By varying the initial concentration of TR fluorophores
incorporated into the SLBs from 0.3% to 0.8% (mol/mol), the maximum
concentration of fluorophores reached during electrophoresis could
be modulated from 2% up to 7% (mol/mol), leading to the reduction
of fluorescence lifetime down to 30% and quenching of the fluorescence
intensity down to 10% of their original levels. As part of this work,
we demonstrated a method for converting fluorescence intensity profiles
into molecular concentration profiles by correcting for quenching
effects. The calculated concentration profiles have a good fit to
an exponential growth function, suggesting that TR-lipids can diffuse
freely even at high concentrations. Overall, these findings prove
that electrophoresis is effective at producing microscale concentration
gradients of a molecule-of-interest and that FLIM is an excellent
approach to interrogate dynamic changes to molecular interactions
via their photophysical state.
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