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Monitoring Proton Exchange and Triplet States with Fluorescence
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimental Biomolecular Physics)
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 [en]
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: urn:nbn:se:kth:diva-10400ISBN: 978-91-7415-304-0 (print)OAI: oai:DiVA.org:kth-10400DiVA: diva2:216731
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
List of papers
1. Monitoring Kinetics of Highly Environment Sensitive States of Fluorescent Molecules by Modulated Excitation and Time-Averaged Fluorescence Intensity Recording
Open this publication in new window or tab >>Monitoring Kinetics of Highly Environment Sensitive States of Fluorescent Molecules by Modulated Excitation and Time-Averaged Fluorescence Intensity Recording
Show others...
2007 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 79, no 9, 3330-3341 p.Article in journal (Refereed) Published
Abstract [en]

In this work, a concept is described for how the kinetics of photoinduced, transient, long-lived, nonfluorescent or weakly fluorescent states of fluorophore marker molecules can be extracted from the time-averaged fluorescence by using time-modulated excitation. The concept exploits the characteristic variation of the population of these states with the modulation parameters of the excitation and thereby circumvents the need for time resolution in the fluorescence detection. It combines the single-molecule sensitivity of fluorescence detection with the remarkable environmental responsiveness obtainable from long-lived transient states, yet does not in itself impose any constraints on the concentration or the fluorescence brightness of the sample molecules that can be measured. Modulation of the excitation can be performed by variation of the intensity of a stationary excitation beam in time or by repeated translations of a CW excitation beam with respect to the sample. As a first experimental verification of the approach, we have shown how the triplet-state parameters of the fluorophore rhodamine 6G in different aqueous enviroments can be extracted. We demonstrate that the concept is fully compatible with low time-resolution detection by a CCD camera. The concept opens for automated transient-state monitoring or imaging on a massively parallel scale and for high-throughput biomolecular screening as well as for more fundamental biomolecular studies. The concept should also be applicable to the monitoring of a range of other photoinduced nonfluorescent or weakly fluorescent transient states, from which subtle changes in the immediate microenvironment of the fluorophore marker molecules can be detected

Keyword
Biomarkers; CCD cameras; Imaging systems; Molecular biology; Sensitivity analysis
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-7194 (URN)10.1021/ac0622680 (DOI)000246027100008 ()2-s2.0-34248153327 (Scopus ID)
Note

QC 20100805

Available from: 2007-05-29 Created: 2007-05-29 Last updated: 2017-12-14Bibliographically approved
2. Transient State Imaging for Microenvironmental Monitoring by Laser Scanning Microscopy
Open this publication in new window or tab >>Transient State Imaging for Microenvironmental Monitoring by Laser Scanning Microscopy
2008 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 80, no 24, 9589-9596 p.Article in journal (Refereed) Published
Abstract [en]

Photoinduced transient dark states are exhibited by practically all common fluorophores. These relatively long-lived states are very sensitive to the local environment and thus highly attractive for microenvironmental imaging purposes. However, because of methodological constraints, their sensitivity has to date been very sparsely exploited. Here, a concept based on spatio-temporal modulation of the excitation intensity is presented that can image these states via their photodynamic fingerprints. With the use of a standard laser scanning microscope, it unites the outstanding environmental sensitivity of the transient state parameters with the high sensitivity of the fluorescence readout and is easily implemented. For demonstration, triplet state images of liposomes with different internal environments were generated. These images provide an example of bow local environmental differences can be resolved, which are not clearly distinguishable via other fluorescence parameters. Having minor instrumental and sample constraints the concept can be foreseen to provide several new, useful, and independent fluorescence-based parameters in biomolecular imaging.

Place, publisher, year, edition, pages
Washington, DC: American Chemical Society, 2008
Keyword
fluorescence correlation spectroscopy; absorption-spectroscopy; phosphorescence; dynamics
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-10236 (URN)10.1021/ac8018735 (DOI)000261728900027 ()2-s2.0-58149234147 (Scopus ID)
Note
QC 20100805Available from: 2009-04-21 Created: 2009-04-21 Last updated: 2017-12-13Bibliographically approved
3. Localized Proton Microcircuits at the Biological Membrane-Water Interface
Open this publication in new window or tab >>Localized Proton Microcircuits at the Biological Membrane-Water Interface
2006 (English)In: PNAS, ISSN 0027-8424, Vol. 103, no 52, 19677-19770 p.Article in journal (Refereed) Published
Abstract [en]

Cellular processes such as nerve conduction, energy metabolism, and import of nutrients into cells all depend on transport of ions across biological membranes through specialized membrane-spanning proteins. Understanding these processes at a molecular level requires mechanistic insights into the interaction between these proteins and the membrane itself. To explore the role of the membrane in ion translocation we used an approach based on fluorescence correlation spectroscopy. Specifically, we investigated exchange of protons between the water phase and the membrane surface, as well as diffusion of protons along membrane surfaces, at a single-molecule level. We show that the lipid head groups collectively act as a proton-collecting antenna, dramatically accelerating proton uptake from water to a membrane-anchored proton acceptor. Furthermore, the results show that proton transfer along the surface can be significantly faster than that between the lipid head groups and the surrounding water phase. Thus, ion translocation across membranes and between the different membrane protein components is a complex interplay between the proteins and the membrane itself, where the membrane acts as a proton-conducting link between membrane-spanning proton transporters

Keyword
diffusion; fluorescence; membrane protein; pH; proton transfer
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-6539 (URN)10.1073/pnas.0605909103 (DOI)000243285500030 ()2-s2.0-33845936297 (Scopus ID)
Note

QC 20100809

Available from: 2006-12-07 Created: 2006-12-07 Last updated: 2016-05-16Bibliographically approved
4. Surface-coupled proton exchange of a membrane-bound proton acceptor
Open this publication in new window or tab >>Surface-coupled proton exchange of a membrane-bound proton acceptor
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
Keyword
biomembrane, diffusion, electrostatic potential, luorescence correlation spectroscopy (FCS), proton transfer
National Category
Analytical Chemistry Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-10399 (URN)10.1073/pnas.0908671107 (DOI)000275131100035 ()2-s2.0-77749239744 (Scopus ID)
Funder
Swedish Research Council, VR-NT-2009-3134Knut and Alice Wallenberg Foundation
Note

Uppdaterad från manuskript till artikel: 20100809 QC 20100809

Available from: 2009-05-11 Created: 2009-05-11 Last updated: 2017-12-13Bibliographically approved
5. Lateral Proton Transfer between the Membrane and a Membrane Protein
Open this publication in new window or tab >>Lateral Proton Transfer between the Membrane and a Membrane Protein
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.

Keyword
cytochrome-c-oxidase; rhodobacter-sphaeroides; molecular-dynamics; cytoplasmic sites; binding sites; surface; bulk; bacteriorhodopsin; diffusion; ph
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-10398 (URN)10.1021/bi8022152 (DOI)000264059500012 ()2-s2.0-64349097112 (Scopus ID)
Note

QC 20100809

Available from: 2009-05-11 Created: 2009-05-11 Last updated: 2017-12-13Bibliographically approved
6. Fluorescence cross-correlation spectroscopy of a pH-sensitive ratiometric dye for molecular proton exchange studies
Open this publication in new window or tab >>Fluorescence cross-correlation spectroscopy of a pH-sensitive ratiometric dye for molecular proton exchange studies
2009 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 11, no 21, 4410-4418 p.Article in journal (Refereed) Published
Abstract [en]

Fluorescence fluctuation analysis of individual pH-sensitive fluorophores has recently proven to be a useful approach for biomolecular proton exchange studies. In this work, dual-color fluorescence cross-correlation spectroscopy (FCCS) is demonstrated on a ratiometric pH-sensitive dye, for which both the excitation and emission spectra shift as a function of pH. In the FCCS measurements, the fluorescence signal from the predominant emission wavelength range of the protonated form of the dye is cross-correlated with that of the deprotonated form. Two lasers are used alternatingly to excite predominantly the protonated and the deprotonated form of the dye. The alternating excitation modulation scheme is combined with detection gating, and is based on a recently developed concept that allows extraction of correlation data for all correlation times regardless of the chosen modulation period. The scheme can thus be applied without concern for the time-scales of the molecular dynamic processes to be studied. By this combined discrimination based on both excitation and emission, spectral cross-talk is dramatically reduced and a very distinct and unambiguous anticorrelation can be recorded in the correlation curves as a consequence of the proton exchange. The strong discrimination power makes the approach applicable also to ratiometric dyes with less pronounced spectral shifts. It should also be useful in combination with ratiometric dyes sensitive to other ambient conditions and ions, such as the biologically very important calcium ion.

Place, publisher, year, edition, pages
Cambridge, UK: RSC Publishing, 2009
Keyword
living cells; single molecules; excitation; microscopy; kinetics
National Category
Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-10239 (URN)10.1039/b822494c (DOI)000266269200035 ()2-s2.0-65949124070 (Scopus ID)
Note
QC 20100805Available from: 2009-04-21 Created: 2009-04-21 Last updated: 2017-12-13Bibliographically approved

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