Studying Ion Exchange in Solution and at Biological Membranes by FCS

Widengren, Jerker

doi:10.1016/b978-0-12-405539-1.00008-7

Cited by 2 publications

(3 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The recorded correlation curves were analyzed using a Levenberg-Marquardt nonlinear least-square curve fitting algorithm (Origin 8; OriginLab, Northampton, MA). A model correlation function was used for the FCS curve fitting, assuming that the fluorescence intensity fluctuations are generated by (1) diffusion (of free fluorophores or of fluorophore-labeled NDs) into and out of the confocal detection volume, and (2) transitions of the fluorophores back and forth into three different dark states ( 19 ):

Here,

is the detected fluorescence intensity at a time t ; τ is the correlation time;

denotes the measurement time over which the fluorescence fluctuations are integrated; square brackets signify the time average; τ D is the average translational diffusion time of the fluorescent species through the confocal detection volume; N is the mean number of fluorophores in the detection volume; and β is the relationship between the axial and lateral extension of the detection volume. The value P signifies the fraction of protonated fluorophores, and τ prot = 1/ k prot is the proton relaxation time, where k prot is the proton relaxation rate.…”

Section: Methodsmentioning

confidence: 99%

“…The recorded correlation curves were analyzed using a Levenberg-Marquardt nonlinear least-square curve fitting algorithm (Origin 8; OriginLab, Northampton, MA). A model correlation function was used for the FCS curve fitting, assuming that the fluorescence intensity fluctuations are generated by ( 1) diffusion (of free fluorophores or of fluorophore-labeled NDs) into and out of the confocal detection volume, and (2) transitions of the fluorophores back and forth into three different dark states (19):…”

Section: Fcs Measurements and Fittingmentioning

confidence: 99%

“…By fluorescence intensity fluctuations studied via FCS, blinking rates and the fractions of fluorescent and nonfluorescent fluorophores can be determined. Monitoring a low number of pH-sensitive fluorophores at a time as they diffuse through the confocal detection volume, free in solution, or labeled to lipids or proteins, it is also possible to determine the proton exchange to and from the fluorophores in their specific local environment at steady state ( 17 , 18 , 19 ). Using this approach, proton uptake rates of dyes bound to a lipid within small (30-nm diameter) 1,2-dileoyl- sn -glycero-3-[phospo- rac -(1-glycerol)] (DOPG) liposomes ( 20 , 21 ), or to the surface of Cyt c O, subsequently inserted into the same type of liposomes ( 22 ), have been studied.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers

Öjemyr

Bergstrand

et al. 2016

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

Lipid membrane surfaces can act as proton-collecting antennae, accelerating proton uptake by membrane-bound proton transporters. We investigated this phenomenon in lipid nanodiscs (NDs) at equilibrium on a local scale, analyzing fluorescence fluctuations of individual pH-sensitive fluorophores at the membrane surface by fluorescence correlation spectroscopy (FCS). The protonation rate of the fluorophores was ∼100-fold higher when located at 9- and 12-nm diameter NDs, compared to when in solution, indicating that the proton-collecting antenna effect is maximal already for a membrane area of ∼60 nm(2). Fluorophore-labeled cytochrome c oxidase displayed a similar increase when reconstituted in 12 nm NDs, but not in 9 nm NDs, i.e., an acceleration of the protonation rate at the surface of cytochrome c oxidase is found when the lipid area surrounding the protein is larger than 80 nm(2), but not when below 30 nm(2). We also investigated the effect of external buffers on the fluorophore proton exchange rates at the ND membrane-water interfaces. With increasing buffer concentrations, the proton exchange rates were found to first decrease and then, at millimolar buffer concentrations, to increase. Monte Carlo simulations, based on a simple kinetic model of the proton exchange at the membrane-water interface, and using rate parameter values determined in our FCS experiments, could reconstruct both the observed membrane-size and the external buffer dependence. The FCS data in combination with the simulations indicate that the local proton diffusion coefficient along a membrane is ∼100 times slower than that observed over submillimeter distances by proton-pulse experiments (Ds ∼ 10(-5)cm(2)/s), and support recent theoretical studies showing that proton diffusion along membrane surfaces is time- and length-scale dependent.

show abstract

Here,

is the detected fluorescence intensity at a time t ; τ is the correlation time;

Section: Methodsmentioning

confidence: 99%

“…The recorded correlation curves were analyzed using a Levenberg-Marquardt nonlinear least-square curve fitting algorithm (Origin 8; OriginLab, Northampton, MA). A model correlation function was used for the FCS curve fitting, assuming that the fluorescence intensity fluctuations are generated by ( 1) diffusion (of free fluorophores or of fluorophore-labeled NDs) into and out of the confocal detection volume, and (2) transitions of the fluorophores back and forth into three different dark states (19):…”

Section: Fcs Measurements and Fittingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers

Öjemyr

Bergstrand

et al. 2016

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

show abstract

Mechanisms of generation of local ΔpH in mitochondria and bacteria

Medvedev

Stuchebrukhov

2014

Biochemistry Moscow

View full text Add to dashboard Cite

The concepts of global and local coupling between proton generators, the enzymes of the respiratory chain, and the consumer, the ATP synthase, coexist in the theory of oxidative phosphorylation. Global coupling is trivial proton transport via the aqueous medium, whereas local coupling implies that the protons pumped are consumed before they escape to the bulk phase. In this work, the conditions for the occurrence of local coupling are explored. It is supposed that the membrane retains protons near its surface and that the proton current generated by the proton pumps rapidly decreases with increasing proton motive force (pmf). It is shown that the competition between the processes of proton translocation across the membrane and their dissipation from the surface to the bulk can result in transient generation of a local ΔpH in reply to a sharp change in pmf; the appearance of local ΔpH, in turn, leads to rapid recovery of the pmf, and hence, it provides for stabilization of the potential at the membrane. Two mechanisms of such kind are discussed: 1) pH changes in the surface area due to proton pumping develop faster than those due to proton escape to the bulk; 2) the former does not take place, but the protons leaving the surface do not equilibrate with the bulk immediately; rather, they give rise to a non-equilibrium concentration near the surface and, as a result, to a back proton flow to the surface. The first mechanism is more efficient, but it does not occur in mitochondria and neutrophilic bacteria, whereas the second can produce ΔpH on the order of unity. In the absence of proton retardation at the surface, local ΔpH does not arise, whereas the formation of global ΔpH is possible only at buffer concentration of less than 10 mM. The role of the mechanisms proposed in transitions between States 3 and 4 of the respiratory chain is discussed. The main conclusion is that surface protons, under conditions where they play a role, support stabilization of the membrane pmf and rapid communication between proton generators and consumers, while their contribution to the energetics is not significant.

show abstract

Studying Ion Exchange in Solution and at Biological Membranes by FCS

Cited by 2 publications

References 49 publications

Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers

Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers

Mechanisms of generation of local ΔpH in mitochondria and bacteria

Contact Info

Product

Resources

About