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Proceedings Paper

"Sizing" the oligomers of Azami Green fluorescent protein with FCS and antibunching
Author(s): Jamshid Temirov; James H. Werner; Peter M. Goodwin; Andrew R. M. Bradbury
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Paper Abstract

Fluorescent proteins are invaluable molecules in fluorescence microscopy and spectroscopy. The size and brightness of fluorescent proteins often dictates the application they may be used for. While a monomeric protein may be the least perturbative structure for labeling a protein in a cell, often oligomers (dimers and tetramers) of fluorescent proteins can be more stable. However, from a quantitative microscopy standpoint, it is important to realize the photophysical properties of monomers do not necessarily multiply by their number when they form oligomers. In this work we studied oligomerization states of the Azami Green (AG) protein with fluorescence correlation spectroscopy (FCS) and photon antibunching or photon pair correlation spectroscopy (PPCS). FCS was used to measure the hydrodynamic size of the oligomers, whereas antibunching was used to count the number of fluorescent emitters in the oligomers. The results exhibited that the dimers of AG were single emitters and the tetramers were dual-emitters, indicative of dipole-dipole interactions and energy transfer between the monomeric units. We also used these methods to estimate the number of fluorescent proteins displayed on T7 phage molecules.

Paper Details

Date Published: 13 February 2012
PDF: 9 pages
Proc. SPIE 8228, Single Molecule Spectroscopy and Superresolution Imaging V, 82280J (13 February 2012); doi: 10.1117/12.906843
Show Author Affiliations
Jamshid Temirov, St. Jude Children's Research Hospital (United States)
James H. Werner, Los Alamos National Lab. (United States)
Peter M. Goodwin, Los Alamos National Lab. (United States)
Andrew R. M. Bradbury, Los Alamos National Lab. (United States)

Published in SPIE Proceedings Vol. 8228:
Single Molecule Spectroscopy and Superresolution Imaging V
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling; Ingo Gregor, Editor(s)

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