"Sizing" the oligomers of Azami Green fluorescent protein with FCS and antibunching

Jamshid Temirov; James H. Werner; Peter M. Goodwin; Andrew R. M. Bradbury

doi:10.1117/12.906843

13 February 2012 "Sizing" the oligomers of Azami Green fluorescent protein with FCS and antibunching

Jamshid Temirov, James H. Werner, Peter M. Goodwin, Andrew R. M. Bradbury

Proceedings Volume 8228, Single Molecule Spectroscopy and Superresolution Imaging V; 82280J (2012) https://doi.org/10.1117/12.906843
Event: SPIE BiOS, 2012, San Francisco, California, United States

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.

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Jamshid Temirov, James H. Werner, Peter M. Goodwin, and Andrew R. M. Bradbury ""Sizing" the oligomers of Azami Green fluorescent protein with FCS and antibunching", Proc. SPIE 8228, Single Molecule Spectroscopy and Superresolution Imaging V, 82280J (13 February 2012); https://doi.org/10.1117/12.906843

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KEYWORDS

Fluorescence correlation spectroscopy

Molecules

Proteins

Fluorescent proteins

Green fluorescent protein

Luminescence

Diffusion

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