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Lateral Proton Transfer between the Membrane and a Membrane Protein
Stockholm University, dept Biochem and Biophys.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.ORCID iD: 0000-0003-3200-0374
Stockholm University, dept Biochem and Biophys.
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 10, 2173-2179 p.Article in journal (Refereed) Published
Abstract [en]

Proton transport across biological membranes is a key step of the energy conservation machinery in living organisms, and it has been proposed that the membrane itself plays an important role in this process. In the present study we have investigated the effect of incorporation of a proton transporter, cytochrome c oxidase, into a membrane on the protonation kinetics of a fluorescent pH-sensitive probe attached at the surface of the protein. The results show that proton transfer to the probe was slightly accelerated upon attachment at the protein surface (similar to 7 x 10(10) s(-1) M-1, compared to the expected value of (1-2) x 10(10) s(-1) M-1), which is presumably due to the presence of acidic/His groups in the vicinity. Upon incorporation of the protein into small unilamellar phospholipid vesicles the rate increased by more than a factor of 400 to similar to 3 x 10(13) s(-1) M-1, which indicates that the protein-attached probe is in rapid protonic contact with the membrane surface. The results indicate that. the membrane acts to accelerate proton uptake by the membrane-bound proton transporter.

Place, publisher, year, edition, pages
2009. Vol. 48, no 10, 2173-2179 p.
Keyword [en]
cytochrome-c-oxidase; rhodobacter-sphaeroides; molecular-dynamics; cytoplasmic sites; binding sites; surface; bulk; bacteriorhodopsin; diffusion; ph
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-10398DOI: 10.1021/bi8022152ISI: 000264059500012Scopus ID: 2-s2.0-64349097112OAI: oai:DiVA.org:kth-10398DiVA: diva2:216702
Note

QC 20100809

Available from: 2009-05-11 Created: 2009-05-11 Last updated: 2017-12-13Bibliographically 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.
Series
Trita-FYS, ISSN 0280-316X ; 2009:14
Keyword
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
Identifiers
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)
Opponent
Supervisors
Note
QC 20100809Available from: 2009-05-13 Created: 2009-05-11 Last updated: 2011-01-24Bibliographically approved

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