SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Quantitative Fluorescence Correlation Spectroscopy reveals a 1000-fold increase in lifetime of protein functionality
Dianwen Zhang 1, Hannes Lans 2, Aufried Lenferink 1, Wim Vermeulen 2 and Cees Otto 1*
1 University of Twente
2 Erasmus Medical Center
* To whom correspondence should be addressed. E-mail: c.otto{at}utwente.nl.
Submitted on March 12, 2008
Revised on April 28, 2008
Accepted on 21 May 2008
 |
Abstract |
|---|
We have investigated dilute protein solutions with fluorescence correlation spectroscopy (FCS) and have observed that a rapid loss of proteins occurs from solution. It is commonly assumed that such a loss is the result of protein adsorption to interfaces. A protocol was developed in which this mode of protein loss can be prevented. However, FCS on fluorescent protein (eGFP, mCherry and mStrawberry) solutions enclosed by adsorption protected interfaces still reveals a decrease of the fluorescent protein concentration, while the diffusion time is stable over long periods of time. We interpret this decay as a loss of protein functionality, probably caused by denaturation of the fluorescent proteins. We show that the typical lifetime of protein functionality in highly dilute, approximately single molecule per femtoliter solutions can be extended more than thousand-fold (typically from a few hours to > 40 days) by adding compounds with surfactant behaviour. No direct interactions between the surfactant and the fluorescent proteins were observed from the diffusion time measured by FCS. A critical surfactant concentration of more than 23 µM was required to achieve the desired protein stabilization for Triton X-100 (TX100). The surfactant does not interfere with DNA-protein binding, because similar observations were made using DNA-cutting restriction enzymes. We associate the occurrence of denaturation of proteins with the activity of water at the water-protein interface which was recently proposed in terms of the "water attack model" (1, 2). Our observations suggest that soluble bio-molecules can extend an influence over much larger distances than suggested by their actual volume.
Key Words:
fluorescence correlation spectroscopy, fluorescent proteins, protein functionality
