Physics Colloquium - Spring 2007 - Development of a Photoactivatable Fluorescent Protein from Aequorea victoria GFP
Dept of Physics & Astronomy
University of Maine, Orono, Maine
Presents
Dr. George Patterson
Cell Biology and Metabolism Branch, NICHD, NIH,
Bethesda Maryland
Development of a Photoactivatable Fluorescent Protein from Aequorea victoria GFP
The green fluorescent protein (GFP) provides a specific and non-invasive fluorescent marker that can be used in a variety of imaging techniques to monitor protein dynamics within living cells. We describe the development of a photoactivatable fluorescent protein based on a T203H mutant of the Aequorea victoria GFP (referred to as PA-GFP). After photoactivation with light of ~400 nm, PA-GFP exhibits a new absorbance peak at ~505 nm that results in a 60-100-fold fluorescence increase over background under 488 nm excitation. The photoactivation occurs in purified protein samples as well as when the proteins are expressed in living cells under aerobic conditions. We have demonstrated the utility of PA-GFP expressed as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein (lgp120) to demonstrate rapid inter-lysosomal membrane exchange. Photoactivation of PA-GFP provides a good complement to the assortment of photobleaching methods currently employed to highlight selectively a pool of fluorescent proteins in a cell. Because only photoactivated molecules exhibit noticeable fluorescence under 488 nm excitation, PA-GFP has the advantage that newly synthesized molecules will not become fluorescent and complicate experimental results. Therefore, these data indicate that PA-GFP will be an excellent tool for biological studies monitoring protein, organelle, and cell dynamics.
Friday, April 6, 2007
3:10 pm
140 Bennett Hall
Refreshments will follow in Rm. 114, Bennett Hall
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