6 research outputs found
Radial Sizing of Lipid Nanotubes Using Membrane Displacement Analysis
We report a novel method for the measurement of lipid
nanotube
radii. Membrane translocation is monitored between two nanotube-connected
vesicles, during the expansion of a receiving vesicle, by observing
a photobleached region of the nanotube. We elucidate nanotube radii,
extracted from SPE vesicles, enabling quantification of membrane composition
and lamellarity. Variances of nanotube radii were measured, showing
a growth of 40–56 nm, upon increasing cholesterol content from
0 to 20%
Probing Structure and Function of Ion Channels Using Limited Proteolysis and Microfluidics
Even though gain, loss, or modulation
of ion channel function is
implicated in many diseases, both rare and common, the development
of new pharmaceuticals targeting this class has been disappointing,
where it has been a major problem to obtain correlated structural
and functional information. Here, we present a microfluidic
method in which the ion channel TRPV1, contained in proteoliposomes
or in excised patches, was exposed to limited trypsin proteolysis.
Cleaved-off peptides were identified by MS, and electrophysiological
properties were recorded by patch clamp. Thus, the structure–function
relationship was evaluated by correlating changes in function with
removal of structural elements. Using this approach, we pinpointed
regions of TRPV1 that affect channel properties upon their removal,
causing changes in current amplitude, single-channel conductance,
and EC<sub>50</sub> value toward its agonist, capsaicin. We have provided
a fast “shotgun” method for chemical truncation of a
membrane protein, which allows for functional assessments of various
peptide regions
Probing Enzymatic Activity Inside Single Cells
We
report a novel approach for determining the enzymatic activity
within a single suspended cell. Using a steady-state microfluidic
delivery device and timed exposure to the pore-forming agent digitonin,
we controlled the plasma membrane permeation of individual NG108-15
cells. Mildly permeabilized cells (∼100 pores) were exposed
to a series of concentrations of fluorescein diphosphate (FDP), a
fluorogenic alkaline phosphatase substrate, with and without levamisole,
an alkaline phosphatase inhibitor. We generated quantitative estimates
for intracellular enzyme activity and were able to construct both
dose-response and dose-inhibition curves at the single-cell level,
resulting in an apparent Michaelis contant <i>K</i><sub>m</sub> of 15.3 μM ± 1.02 (mean ± standard error
of the mean (SEM), <i>n</i> = 16) and an inhibition constant <i>K</i><sub>i</sub> of 0.59 mM ± 0.07 (mean ± SEM, <i>n</i> = 14). Enzymatic activity could be monitored just 40 s
after permeabilization, and five point dose-inhibition curves could
be obtained within 150 s. This rapid approach offers a new methodology
for characterizing enzyme activity within single cells
Kinetics of Diffusion-Mediated DNA Hybridization in Lipid Monolayer Films Determined by Single-Molecule Fluorescence Spectroscopy
We use single-molecule fluorescence microscopy to monitor individual hybridization reactions between membrane-anchored DNA strands, occurring in nanofluidic lipid monolayer films deposited on Teflon AF substrates. The DNA molecules are labeled with different fluorescent dyes, which make it possible to simultaneously monitor the movements of two different molecular species, thus enabling tracking of both reactants and products. We employ lattice diffusion simulations to determine reaction probabilities upon interaction. The observed hybridization rate of the 40-mer DNA was more than 2-fold higher than that of the 20-mer DNA. Since the lateral diffusion coefficient of the two different constructs is nearly identical, the effective molecule radius determines the overall kinetics. This implies that when two DNA molecules approach each other, hydrogen bonding takes place distal from the place where the DNA is anchored to the surface. Strand closure then propagates bidirectionally through a zipper-like mechanism, eventually bringing the lipid anchors together. Comparison with hybridization rates for corresponding DNA sequences in solution reveals that hybridization rates are lower for the lipid-anchored strands and that the dependence on strand length is stronger
Kinetics of Diffusion-Mediated DNA Hybridization in Lipid Monolayer Films Determined by Single-Molecule Fluorescence Spectroscopy
We use single-molecule fluorescence microscopy to monitor individual hybridization reactions between membrane-anchored DNA strands, occurring in nanofluidic lipid monolayer films deposited on Teflon AF substrates. The DNA molecules are labeled with different fluorescent dyes, which make it possible to simultaneously monitor the movements of two different molecular species, thus enabling tracking of both reactants and products. We employ lattice diffusion simulations to determine reaction probabilities upon interaction. The observed hybridization rate of the 40-mer DNA was more than 2-fold higher than that of the 20-mer DNA. Since the lateral diffusion coefficient of the two different constructs is nearly identical, the effective molecule radius determines the overall kinetics. This implies that when two DNA molecules approach each other, hydrogen bonding takes place distal from the place where the DNA is anchored to the surface. Strand closure then propagates bidirectionally through a zipper-like mechanism, eventually bringing the lipid anchors together. Comparison with hybridization rates for corresponding DNA sequences in solution reveals that hybridization rates are lower for the lipid-anchored strands and that the dependence on strand length is stronger
Microfluidic Flow Cell for Sequential Digestion of Immobilized Proteoliposomes
We have developed a microfluidic flow cell where stepwise
enzymatic
digestion is performed on immobilized proteoliposomes and the resulting
cleaved peptides are analyzed with liquid chromatography–tandem
mass spectrometry (LC–MS/MS). The flow cell channels consist
of two parallel gold surfaces mounted face to face with a thin spacer
and feature an inlet and an outlet port. Proteoliposomes (50–150
nm in diameter) obtained from red blood cells (RBC), or Chinese hamster
ovary (CHO) cells, were immobilized on the inside of the flow cell
channel, thus forming a stationary phase of proteoliposomes. The rate
of proteoliposome immobilization was determined using a quartz crystal
microbalance with dissipation monitoring (QCM-D) which showed that
95% of the proteoliposomes bind within 5 min. The flow cell was found
to bind a maximum of 1 μg proteoliposomes/cm<sup>2</sup>, and
a minimum proteoliposome concentration required for saturation of
the flow cell was determined to be 500 μg/mL. Atomic force microscopy
(AFM) studies showed an even distribution of immobilized proteoliposomes
on the surface. The liquid encapsulated between the surfaces has a
large surface-to-volume ratio, providing rapid material transfer rates
between the liquid phase and the stationary phase. We characterized
the hydrodynamic properties of the flow cell, and the force acting
on the proteoliposomes during flow cell operation was estimated to
be in the range of 0.1–1 pN, too small to cause any proteoliposome
deformation or rupture. A sequential proteolytic protocol, repeatedly
exposing proteoliposomes to a digestive enzyme, trypsin, was developed
and compared with a single-digest protocol. The sequential protocol
was found to detect ∼65% more unique membrane-associated protein
(<i>p</i> < 0.001, <i>n</i> = 6) based on peptide
analysis with LC–MS/MS, compared to a single-digest protocol.
Thus, the flow cell described herein is a suitable tool for shotgun
proteomics on proteoliposomes, enabling more detailed characterization
of complex protein samples