PNNL-13501 - Pacific Northwest National Laboratory

laboratory (Lu and Xie 1997; Lu et al. 1998; Xie and Lu
1999) and others (Xie and Trautman 1998) demonstrated
that single-molecule fluorescence spectrum and lifetime
typically fluctuate as the changes of local environment
and internal or external conformations of proteins.
Using a fast, high-sensitivity, intensified charge coupled
device camera and our time-correlated single-photon
counting trajectory device, we can record the spectral and
fluorescence lifetime fluctuation trajectories. These
reveal intrinsic single-molecule conformational dynamics
and environmental change dynamics by calculating first
and higher order autocorrelation functions from the
trajectories. Single-pair fluorescence resonant energy
transfer measures the distance change with angstrom-level
sensitivity between a single donor/acceptor pair in a
single-protein or single-complex. Molecule
conformational changes, protein motions, ligand-protein
and protein-protein interactions can be monitored by
exciting the donor and monitoring either the donor’s and
acceptor’s intensity trajectories or both, but separately.
Gramicidin channels (Figure 2) are chosen as a model ion
channel for the system development because of their
known structure, well-defined function, ease of
preparation, and commercial availability. A gramicidin
channel is a dimer of two 15-amino acid units that meet
head-to-head in the interior of the membrane bilayer.
Cations, but not anions, permeate through the resulting
structure. There is a wealth of single-channel
electrophysiological data for permeation through the
channel in different lipid types and with different salt
concentrations. However, single-gramicidin-channel
conformational changes, correlated with ion channel
current trajectories, have never been recorded. The
knowledge of how the channels work in real-time is
absent.
Results and Accomplishments
We have designed, developed, and installed a patchclamp
instrumental component coupled with a real-time
scanning confocal microscope, establishing a formal
procedure to make the patch-clamp pipette tips. Singlepeptide
ion channel current trajectories at the picoampere
level were recorded with low noise (less than 0.5
picoampere), and statistical analysis programs were
developed to analyze the stochastic ion channel openclose
kinetics. The dimerization dynamics of singlepeptide
ion channels, gramicidin A and dansyl gramicidin
C, were probed by monitoring single-ion channel current
on and off trajectories, and the dynamics were found to be
inhomogeneous due to different local environments
around the single-dimers and monomers. Extensive
exploratory experiments have been carried out in order to
obtain fluorescence single-ion channel imaging and
correlate dimerization dynamics measurements from both
fluorescence trajectories and ion current trajectories using
the combined patch-clamp confocal microscope. These
results may be summarized as follows:
1. We designed, developed, and installed a patch-clamp
instrumental component coupled with a real-time
scanning confocal microscope.
2. We developed and improved the procedures of
preparing patch-clamp micropipette tips with defined
aperture sizes (5-µm to 500-nm in diameter).
3. We developed and improved the technical methods of
preparing stable lipid vesicles and lipid bilayers
formed at the patch-clamp pipette tips.
4. We achieved measurements of single-ion channel
(gramicidin dimers) current (on-off) trajectories with
a high sensitivity (pA) (Figure 2).
5. We established computational data analysis of singleion
channel stochastic time trajectories (Figure 3).
6. We designed, developed, and installed a singlemolecule
optical imaging instrumental component
coupled with the real-time scanning confocal
microscope and the patch-clamp instrument. Using a
galvo-mirror two-dimensional scanning mechanism,
we were able to scan a laser diffraction-limited
focused spot using either cw laser excitation for
linear fluorescence imaging and femtosecond pulse
laser for two-photon fluorescence imaging. Twophoton
fluorescence imaging gives high spatial
resolution (beyond diffraction limit) and low
autofluorescence background.
7. We installed and tested an intensified change coupled
device camera that has single-photon counting
sensitivity and video-rate imaging collection speed.
Installed computer-controlled two-dimensional
electropiezo close-loop scanning stage with nmprecision
to facilitate focusing laser on a selected
single-ion channel protein under a patch-clamp
pipette aperture.
8. We conducted extensive single-molecule imaging
exploratory experiments.
Biosciences and Biotechnology 33