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Lippitz, Markus ; Erker, Wolfgang ; Decker, Heinz ; van Holde, K. E. ; Basche, Thomas:
Two-photon excitation microscopy of tryptophan-containing proteins.
In: Proceedings of the National Academy of Sciences of the United States of America.
Bd. 99
(2002)
Heft 5
.
- S. 2772-2777.
ISSN 1091-6490
DOI: https://doi.org/10.1073/pnas.052662999
Abstract
We have examined the feasibility of observing single protein molecules by means of their intrinsic tryptophan emission after two-photon excitation. A respiratory protein from spiders, the 24-meric hemocyanin, containing 148 tryptophans, was studied in its native state under almost in vivo conditions. In this specific case, the intensity of the tryptophan emission signals the oxygen load, allowing one to investigate molecular cooperativity. As a system with even higher tryptophan content, we also investigated latex spheres covered with the protein avidin, resulting in 340 tryptophans per sphere. The ratio of the fluorescence quantum efficiency to the bleaching efficiency was found to vary between 2 and 180 after two-photon excitation for tryptophan free in buffer solution, in hemocyanin, and in avidin-coated spheres. In the case of hemocyanin, this ratio leads to about four photons detected before photobleaching. Although this number is quite small, the diffusion of individual protein molecules could be detected by fluorescence correlation spectroscopy. In avidin-coated spheres, the tryptophans exhibit a higher photostability, so that even imaging of single spheres becomes possible. As an unexpected result of the measurements, it was discovered that the population of the oxygenated state of hemocyanin can be changed by means of a one-photon process with the same laser source that monitors this population in a two-photon process.
Weitere Angaben
| Publikationsform: | Artikel in einer Zeitschrift |
|---|---|
| Begutachteter Beitrag: | Ja |
| Institutionen der Universität: | Fakultäten Fakultäten > Fakultät für Mathematik, Physik und Informatik Fakultäten > Fakultät für Mathematik, Physik und Informatik > Physikalisches Institut Fakultäten > Fakultät für Mathematik, Physik und Informatik > Physikalisches Institut > Lehrstuhl Experimentalphysik III - Nanooptik Fakultäten > Fakultät für Mathematik, Physik und Informatik > Physikalisches Institut > Lehrstuhl Experimentalphysik III - Nanooptik > Lehrstuhl Experimentalphysik III - Nanooptik - Univ.-Prof. Dr. Markus Lippitz |
| Titel an der UBT entstanden: | Nein |
| Themengebiete aus DDC: | 500 Naturwissenschaften und Mathematik 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften 500 Naturwissenschaften und Mathematik > 530 Physik |
| Eingestellt am: | 17 Dec 2015 08:17 |
| Letzte Änderung: | 25 Jan 2022 13:44 |
| URI: | https://eref.uni-bayreuth.de/id/eprint/28975 |