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Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
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2016 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 9, p. 1993-2003Article in journal (Refereed) Published
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Text
Abstract [en]

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 similar to 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 similar to 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 similar to 100 times slower than that observed over submillimeter distances by proton-pulse experiments (D-s similar to 10(-5)cm(2)/s), and support recent theoretical studies showing that proton diffusion along membrane surfaces is time- and length-scale dependent.

Place, publisher, year, edition, pages
2016. Vol. 110, no 9, p. 1993-2003
National Category
Biophysics
Identifiers
URN: urn:nbn:se:kth:diva-188060DOI: 10.1016/j.bpj.2016.03.035ISI: 000375896400009Scopus ID: 2-s2.0-84966309427OAI: oai:DiVA.org:kth-188060DiVA, id: diva2:943047
Note

QC 20160627

Available from: 2016-06-27 Created: 2016-06-03 Last updated: 2019-04-04Bibliographically approved
In thesis
1. Super resolution fluorescence imaging: analyses, simulations and applications
Open this publication in new window or tab >>Super resolution fluorescence imaging: analyses, simulations and applications
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence methods offer extraordinary sensitivity and specificity, and are extensively used in the life sciences. In recent years, super resolution fluorescence imaging techniques have developed strongly, uniquely combining ~10 nm sub diffraction resolution and specific labeling with high efficiency. This thesis explores this potential, with a major focus on Stimulated Emission Depletion, STED, microscopy, applications thereof, image analyses and simulation studies. An additional theme in this thesis is development and use of single molecule fluorescence correlation spectroscopy, FCS, and related techniques, as tools to study dynamic processes at the molecular level. In paper I the proteins cytochrome-bo3 and ATP-synthase are studied with fluorescence cross-correlation spectroscopy, FCCS. These two proteins are a part of the energy conversion process in E. coli, converting ADP into ATP. We found that an increased interaction between these proteins, detected by FCCS, correlates with an increase in the ATP production. In paper II an FCS-based imaging method is developed, capable to determine absolute sizes of objects, smaller than the resolution limit of the microscope used. Combined with STED, this may open for studies of membrane nano-domains, such as those investigated by simulations in paper VII. In paper III and paper IV super resolution STED imaging was applied on Streptococcus Pneumoniae, revealing information about function and distribution of proteins involved in the defense mechanism of the bacteria, as well as their role in bacterial meningitis. In paper V, we used STED imaging to investigate protein distributions in platelets. We then found that the adhesion protein P-selectin changes its distribution pattern in platelets incubated with tumor cells, and with machine learning algorithms and classical image analysis of the STED images it is possible to automatically distinguish such platelets from platelets activated by other means. This could provide a strategy for minimally invasive diagnostics of early cancer development, and deeper understanding of the role of platelets in cancer development. Finally, this thesis presents Monte-Carlo simulations of biological processes and their monitoring by FCS. In paper VI, a combination of FCCS and simulations was applied to resolve the interactions between a transcription factor (p53) and an oncoprotein (MDM2) inside live cells. In paper VII, the feasibility of FCS techniques for studying nano-domains in membranes is investigated purely by simulations, identifying the conditions under which such nano-domains would be possible to detect by FCS. In paper VIII, proton exchange dynamics at biological membranes were simulated in a model, verifying experimental FCS data and identifying fundamental mechanisms by which membranes mediate proton exchange on a local (~10nm) scale.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. p. 81
Series
TRITA-SCI-FOU ; 2019:20
National Category
Other Physics Topics
Research subject
Physics
Identifiers
urn:nbn:se:kth:diva-248297 (URN)978-91-7873-171-8 (ISBN)
Public defence
2019-04-26, FA32, KTH, Roslagstullsbacken 21, Stockholm, 18:22 (English)
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Supervisors
Note

QC 20190405

Available from: 2019-04-05 Created: 2019-04-04 Last updated: 2019-04-05Bibliographically approved

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