3 research outputs found
Fluorescence Microscopy of Single Liposomes with Incorporated Pigment-Proteins
Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency, and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here, we present a novel strategy employing methods of proteoliposome preparation, fluorescent labeling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the singleliposome level and for the first time apply it to study photosynthetic pigment protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of nonreconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled nativelike environment
Probing Activation and Conformational Dynamics of the Vesicle-Reconstituted β<sub>2</sub> Adrenergic Receptor at the Single-Molecule Level
G-protein-coupled receptors (GPCRs) are structurally
flexible membrane
proteins that mediate a host of physiological responses to extracellular
ligands like hormones and neurotransmitters. Fine features of their
dynamic structural behavior are hypothesized to encode the functional
plasticity seen in GPCR activity, where ligands with different efficacies
can direct the same receptor toward different signaling phenotypes.
Although the number of GPCR crystal structures is increasing, the
receptors are characterized by complex and poorly understood conformational
landscapes. Therefore, we employed a fluorescence microscopy assay
to monitor conformational dynamics of single β2 adrenergic
receptors (β2ARs). To increase the biological relevance
of our findings, we decided not to reconstitute the receptor in detergent
micelles but rather lipid membranes as proteoliposomes. The conformational
dynamics were monitored by changes in the intensity of an environmentally
sensitive boron-dipyrromethene (BODIPY 493/503) fluorophore conjugated
to an endogenous cysteine (located at the cytoplasmic end of the sixth
transmembrane helix of the receptor). Using total internal reflection
fluorescence microscopy (TIRFM) and a single small unilamellar liposome
assay that we previously developed, we followed the real-time dynamic
properties of hundreds of single β2ARs reconstituted
in a native-like environmentlipid membranes. Our results showed
that β2AR-BODIPY fluctuates between several states
of different intensity on a time scale of seconds, compared to BODIPY-lipid
conjugates that show almost entirely stable fluorescence emission
in the absence and presence of the full agonist BI-167107. Agonist
stimulation changes the β2AR dynamics, increasing
the population of states with higher intensities and prolonging their
durations, consistent with bulk experiments. The transition density
plot demonstrates that β2AR-BODIPY, in the absence
of the full agonist, interconverts between states of low and moderate
intensity, while the full agonist renders transitions between moderate
and high-intensity states more probable. This redistribution is consistent
with a mechanism of conformational selection and is a promising first
step toward characterizing the conformational dynamics of GPCRs embedded
in a lipid bilayer
On Solving the Initial Problem of LR Arrays
Heme-copper oxidases (HCOs) are key
enzymes in prokaryotes and
eukaryotes for energy production during aerobic respiration. They
catalyze the reduction of the terminal electron acceptor, oxygen,
and utilize the Gibbs free energy to transport protons across a membrane
to generate a proton (ΔpH) and electrochemical gradient termed
proton motive force (PMF), which provides the driving force for the
adenosine triphosphate (ATP) synthesis. Excessive PMF is known to
limit the turnover of HCOs, but the molecular mechanism of this regulatory
feedback remains relatively unexplored. Here we present a single-enzyme
study that reveals that cytochrome <i>bo</i><sub>3</sub> from <i>Escherichia coli</i>, an HCO closely homologous
to Complex IV in human mitochondria, can enter a rare, long-lifetime
leak state during which proton flow is reversed. The probability of
entering the leak state is increased at higher ΔpH. By rapidly
dissipating the PMF, we propose that this leak state may enable cytochrome <i>bo</i><sub>3</sub>, and possibly other HCOs, to maintain a suitable
ΔpH under extreme redox conditions