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Surface-coupled proton exchange of a membrane-bound proton acceptor
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
2010 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 9, 4129-4134 p.Article in journal (Refereed) Published
Abstract [en]

Proton-transfer reactions across and at the surface of biological membranes are central for maintaining the transmembrane proton electrochemical gradients involved in cellular energy conversion. In this study, fluorescence correlation spectroscopy was used to measure the local protonation and deprotonation rates of single pH-sensitive fluorophores conjugated to liposome membranes, and the dependence of these rates on lipid composition and ion concentration. Measurements of proton exchange rates over a wide proton concentration range, using two different pH-sensitive fluorophores with different pKas, revealed two distinct proton exchange regimes. At high pH (> 8), proton association increases rapidly with increasing proton concentrations, presumably because the whole membrane acts as a proton-collecting antenna for the fluorophore. In contrast, at low pH (< 7), the increase in the proton association rate is slower and comparable to that of direct protonation of the fluorophore from the bulk solution. In the latter case, the proton exchange rates of the two fluorophores are indistinguishable, indicating that their protonation rates are determined by the local membrane environment. Measurements on membranes of different surface charge and at different ion concentrations made it possible to determine surface potentials, as well as the distance between the surface and the fluorophore. The results from this study define the conditions under which biological membranes can act as proton-collecting antennae and provide fundamental information on the relation between the membrane surface charge density and the local proton exchange kinetics.

Place, publisher, year, edition, pages
Washington: National Academy of Sciences , 2010. Vol. 107, no 9, 4129-4134 p.
Keyword [en]
biomembrane, diffusion, electrostatic potential, luorescence correlation spectroscopy (FCS), proton transfer
National Category
Analytical Chemistry Biophysics
Research subject
Biological Physics
URN: urn:nbn:se:kth:diva-10399DOI: 10.1073/pnas.0908671107ISI: 000275131100035ScopusID: 2-s2.0-77749239744OAI: diva2:216711
Swedish Research Council, VR-NT-2009-3134Knut and Alice Wallenberg Foundation

Uppdaterad från manuskript till artikel: 20100809 QC 20100809

Available from: 2009-05-11 Created: 2009-05-11 Last updated: 2016-03-10Bibliographically approved
In thesis
1. Monitoring Proton Exchange and Triplet States with Fluorescence
Open this publication in new window or tab >>Monitoring Proton Exchange and Triplet States with Fluorescence
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescent molecules commonly shift to transient dark states, induced bylight or triggered by chemical reactions. The transient dark states can beused as probes of the local environment surrounding the fluorescent molecules,and are therefore attractive for use in biomolecular applications. Thisthesis explores the use and development of novel fluorescence spectroscopictechniques for monitoring transient dark states.This work demonstrates that kinetic information regarding photoinduced transient dark states of fluorescent molecules can be obtained from the time-averaged fluorescence intensity of fluorescent molecules subject totemporally modulated illumination. Methods based on this approach havethe advantage that the light detectors can have a low time resolution, which allows for parallelization and screening of biomolecular interactions withhigh throughput. Transient state images are presented displaying local environmental differences such as those in oxygen concentration and quencher accessibility.Analysis of the fluorescence intensity fluctuations resulting from thetransitions to and from transient dark states can be used to obtain information regarding the transition rates and occupancy of the transient darkstates. Fluorescence fluctuation analysis was used to reveal rates of protonbinding and debinding to single fluorescent molecules located close to biological membranes and protein surfaces. The results from these studies show that the proton exchange rate increases dramatically when the fluorescent molecule is close to the membrane.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. vii, 79 p.
Trita-FYS, ISSN 0280-316X ; 2009:14
fluorescence correlation spectroscopy, proton transfer, cytochrome c oxidase, transient state imaging, modulated excitation
National Category
Atom and Molecular Physics and Optics Biochemistry and Molecular Biology
urn:nbn:se:kth:diva-10400 (URN)978-91-7415-304-0 (ISBN)
Public defence
2009-05-15, Sal FB42, AlbaNova, Roslagstullsbacken 21, Sstockholm, 09:00 (English)
QC 20100809Available from: 2009-05-13 Created: 2009-05-11 Last updated: 2011-01-24Bibliographically approved

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