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The membrane proton collecting antenna effect studied by Fluorescence Correlation Spectroscopy in a lipid-nanodisc model system – influence of the membrane area and external buffers
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
Stockholm University, Department of Biochemistry and Biophysics.
Stockholm University, Department of Biochemistry and Biophysics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Lipid membranes can act as proton collecting antennae, thereby significantly accelerating the protonation uptake of proteins responsible for proton transport across biological membranes. This uptake is a crucial step in the energy conversion within all living cells. In this study, we investigated the membrane-size dependence of the proton collecting antenna effect in a lipid nanodisc membrane model system of different sizes, 9nm and 12nm in diameter, with and without incorporation of a proton transporter, cytochrome c oxidase (CytcO). We also investigated how the proton exchange at the membrane-water interface is influenced by buffer molecules in the bulk solution. The proton exchange was monitored via fluorescence fluctuations of individual pH-sensitive fluorophores attached to membranes or to CytcO incorporated into the nanodiscs using fluorescence correlation spectroscopy. Our data confirm the significance of the membrane-water interface for accelerating proton uptake, show that this acceleration depends on the size of the membrane area surrounding the dyes, and indicate that the proton collection antenna can be in operation over a planar membrane-water interface in the range of 10nm in diameter, or possible larger. The buffer dependence for membrane-bound protonatable compounds was found to strongly deviate from the linear dependence, previously observed in both purely three-dimensional and two-dimensional systems. This, more complex, buffer concentration dependence can be explained by considering that also the proton exchange between the membrane surface itself and the bulk is influenced at the different buffer concentrations. Taken together, our findings reveal important biologically relevant aspects for how proton exchange at and across biological membranes are mediated and motivate further studies to better understand how these mechanisms mediate the proton exchange in more complex environments, as experienced in a living cells.

Keyword [en]
protonation, proton collecting antenna effect, nanodisc, Fluorescence Correlation Spectroscopy (FCS)
National Category
Physical Sciences Biophysics
URN: urn:nbn:se:kth:diva-146175OAI: diva2:722593

QS 2014

Available from: 2014-06-09 Created: 2014-06-09 Last updated: 2014-06-09Bibliographically approved
In thesis
1. Development and application of ultra-sensitive fluorescence spectroscopy and microscopy for biomolecular interaction studies
Open this publication in new window or tab >>Development and application of ultra-sensitive fluorescence spectroscopy and microscopy for biomolecular interaction studies
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the development of sensitive and high-resolution fluorescence spectroscopic and microscopic techniques and their application to probe biomolecules and their interactions in solution, lipid membrane model systems and in cells. Paper I-IV are largely focused on methodological developments. In paper I, a new fluorescence method based on fluorescence correlation spectroscopy (FCS) for detecting single particles was realized, requiring no fluorescent labeling of the particles. The method can yield information both about the diffusion properties of the particles as well as about their volumes. In paper II, a modified fluorescence cross correlation spectroscopy procedure with well characterized instrumental calibration was developed and applied to study cis interactions between an inhibitory receptor and its Major Histocompatibility Complex class I ligand molecule, both within the same cellular membranes. The quantitative analysis brought new insights into the Nature killer cell’s self-regulating of tolerance and aggressiveness for immune responses. Paper III describes a multi-color STED (STimulated Emission Depletion) microscopy procedure, capable of imaging four different targets in the same cells at 40nm optical resolution, which was developed and successfully demonstrated on platelets. In paper IV, a modified co-localization algorithm for fluorescence images analysis was proposed, which is essentially insensitive to resolutions and molecule densities. Further, the performance of this algorithm and of using STED microscopy for co-localization analysis was evaluated using both simulated and experimentally acquired images.

Papers V-VII have their main emphasis on the application side. In paper V, transient state imaging was demonstrated on live cells to image intracellular oxygen concentration and successfully differentiated different breast cancer cell lines and the different metabolic pathways they adopted to under different culturing conditions. Paper VI describes a FCS-based study of proton exchange at biological membranes, the size-dependence of the membrane proton collecting antenna effect as well as effects of external buffer solutions on the proton exchange, in a nanodisc lipid membrane model system. These findings provide insights for understanding proton transport at and across membranes of live cells, which has a central biological relevance. In paper VII, STED imaging and co-localization analysis was applied to analyze cell adhesion related protein interactions, which are believed to have an important modulating role for the proliferation, differentiation, survival and motility of the cells. The outcome of efforts taken to develop means for early cancer diagnosis are also presented. It is based on single cells extracted by fine needle aspiration and the use of multi-parameter fluorescence detection and STED imaging to detect protein interactions in the clinical samples. Taken together, detailed studies at a molecular level are critical to understand complex systems such as living organisms. It is the hope that the methodologies developed and applied in this thesis can contribute not only to the development of fundamental science, but also that they can be of benefit to mankind in the field of biomedicine, especially with an ultimate goal of developing novel techniques for cancer diagnosis.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. xv, 79 p.
TRITA-FYS, ISSN 0280-316X ; 2014:23
single molecule spectroscopy, fluorescence correlation spectroscopy, stimulated emission microscopy, cancer, biomolecular interaction, co-localization
National Category
Physical Sciences
Research subject
Biological Physics
urn:nbn:se:kth:diva-146181 (URN)978-91-7595-180-5 (ISBN)
Public defence
2014-06-10, FB42, AlbaNova Universititetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)

QC 20140609

Available from: 2014-06-09 Created: 2014-06-09 Last updated: 2015-06-01Bibliographically approved

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