34 research outputs found
Targeting cytochrome C oxidase in mitochondria with Pt(II)-porphyrins for Photodynamic Therapy
Mitochondria are the power house of living cells, where the synthesis of the
chemical "energy currency" adenosine triphosphate (ATP) occurs. Oxidative
phosphorylation by a series of membrane protein complexes I to IV, that is, the
electron transport chain, is the source of the electrochemical potential
difference or proton motive force (PMF) of protons across the inner
mitochondrial membrane. The PMF is required for ATP production by complex V of
the electron transport chain, i.e. by FoF1-ATP synthase. Destroying cytochrome
C oxidase (COX; complex IV) in Photodynamic Therapy (PDT) is achieved by the
cationic photosensitizer Pt(II)-TMPyP. Electron microscopy revealed the
disruption of the mitochondrial christae as a primary step of PDT. Time
resolved phosphorescence measurements identified COX as the binding site for
Pt(II)-TMPyP in living HeLa cells. As this photosensitizer competed with
cytochrome C in binding to COX, destruction of COX might not only disturb ATP
synthesis but could expedite the release of cytochrome C to the cytosol
inducing apoptosis.Comment: 11 pages, 5 figure
Monitoring transient elastic energy storage within the rotary motors of single FoF1-ATP synthase by DCO-ALEX FRET
The enzyme FoF1-ATP synthase provides the 'chemical energy currency'
adenosine triphosphate (ATP) for living cells. Catalysis is driven by
mechanochemical coupling of subunit rotation within the enzyme with
conformational changes in the three ATP binding sites. Proton translocation
through the membrane-bound Fo part of ATP synthase powers a 10-step rotary
motion of the ring of c subunits. This rotation is transmitted to the gamma and
epsilon subunits of the F1 part. Because gamma and epsilon subunits rotate in
120 deg steps, we aim to unravel this symmetry mismatch by real time monitoring
subunit rotation using single-molecule Forster resonance energy transfer
(FRET). One fluorophore is attached specifically to the F1 motor, another one
to the Fo motor of the liposome-reconstituted enzyme. Photophysical artifacts
due to spectral fluctuations of the single fluorophores are minimized by a
previously developed duty cycle-optimized alternating laser excitation scheme
(DCO-ALEX). We report the detection of reversible elastic deformations between
the rotor parts of Fo and F1 and estimate the maximum angular displacement
during the load-free rotation using Monte Carlo simulationsComment: 14 pages, 7 figure
Regulatory assembly of the vacuolar proton pump VOV1-ATPase in yeast cells by FLIM-FRET
We investigate the reversible disassembly of VOV1-ATPase in life yeast cells
by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane
pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary
biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is
used for transport processes as well as for pH and Ca2+ homoeostasis in the
cell. Activity of the VOV1-ATPase is regulated through assembly / disassembly
processes. During starvation the two parts of VOV1-ATPase start to disassemble.
This process is reversed after addition of glucose. The exact mechanisms are
unknown. To follow the disassembly / reassembly in vivo we tagged two subunits
C and E with different fluorescent proteins. Cellular distributions of C and E
were monitored using a duty cycle-optimized alternating laser excitation scheme
(DCO-ALEX) for time resolved confocal FRET-FLIM measurements.Comment: 8 pages, 3 figure
Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer
In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using
single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of
the ATP binding cassette family of transport proteins, is found in the plasma
membrane of animal cells where it is involved in the ATP hydrolysis driven
export of hydrophobic molecules. When expressed in the plasma membrane of
cancer cells, the transport activity of Pgp can lead to the failure of
chemotherapy by excluding the mostly hydrophobic drugs from the interior of the
cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples
MgATP binding and hydrolysis to translocation of drug molecules across the
lipid bilayer is poorly understood. Using site directed mutagenesis, we have
introduced cysteine residues for fluorescence labeling into different regions
of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp
molecules showed fluctuating FRET efficiencies during drug stimulated ATP
hydrolysis suggesting that the NBDs undergo significant movements during
catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is
applied to minimize FRET artifacts and to select the appropriate molecules. The
data show that Pgp is a highly dynamic enzyme that appears to fluctuate between
at least two major conformations during steady state turnover.Comment: 10 pages, 7 figure
Diffusion properties of single FoF1-ATP synthases in a living bacterium unraveled by localization microscopy
FoF1-ATP synthases in Escherichia coli (E. coli) bacteria are membrane-bound
enzymes which use an internal proton-driven rotary double motor to catalyze the
synthesis of adenosine triphosphate (ATP). According to the 'chemiosmotic
hypothesis', a series of proton pumps generate the necessary pH difference plus
an electric potential across the bacterial plasma membrane. These proton pumps
are redox-coupled membrane enzymes which are possibly organized in
supercomplexes, as shown for the related enzymes in the mitochondrial inner
membrane. We report diffusion measurements of single fluorescent FoF1-ATP
synthases in living E. coli by localization microscopy and single enzyme
tracking to distinguish a monomeric enzyme from a supercomplex-associated form
in the bacterial membrane. For quantitative mean square displacement (MSD)
analysis, the limited size of the observation area in the membrane with a
significant membrane curvature had to be considered. The E. coli cells had a
diameter of about 500 nm and a length of about 2 to 3 \mum. Because the surface
coordinate system yielded different localization precision, we applied a
sliding observation window approach to obtain the diffusion coefficient D =
0.072 \mum2/s of FoF1-ATP synthase in living E. coli cells.Comment: 12 pages, 6 figure
Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET
Thermophilic enzymes can operate at higher temperatures but show reduced
activities at room temperature. They are in general more stable during
preparation and, accordingly, are considered to be more rigid in structure.
Crystallization is often easier compared to proteins from bacteria growing at
ambient temperatures, especially for membrane proteins. The ATP-producing
enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum
strain TA2.A1 is driven by a Fo motor consisting of a ring of 13 c-subunits. We
applied a single-molecule F\"orster resonance energy transfer (FRET) approach
using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor
the expected 13-stepped rotary Fo motor at work. New FRET transition histograms
were developed to identify the smaller step sizes compared to the 10-stepped Fo
motor of the Escherichia coli enzyme. Dwell time analysis revealed the
temperature and the LDAO dependence of the Fo motor activity on the single
molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating
a high flexibility in the membrane part of this thermophilic enzyme.Comment: 14 pages, 7 figure