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Quantitative Fluorescence Correlation Spectroscopy Reveals a 1000-Fold Increase in Lifetime of Protein Functionality
- Source :
- Biophysical Journal, 95(7), 3439-3446. Cell Press, Biophysical journal, 95(7), 3439-3446. Biophysical Society
- Publication Year :
- 2008
- Publisher :
- Elsevier BV, 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 (enhanced green fluorescent protein, 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 1000-fold (typically from a few hours to >40 days) by adding compounds with surfactant behavior. 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 23μM was required to achieve the desired protein stabilization for Triton X-100. 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”. Our observations suggest that soluble biomolecules can extend an influence over much larger distances than suggested by their actual volume.
- Subjects :
- Time Factors
Surface Properties
Chemistry
Biophysics
Analytical chemistry
Proteins
Water
Fluorescence correlation spectroscopy
DNA
DNA Restriction Enzymes
Fluorescence
Surface-Active Agents
Spectrometry, Fluorescence
Spectroscopy, Imaging, Other Techniques
Pulmonary surfactant
Animals
Cattle
Mutant Proteins
Denaturation (biochemistry)
Adsorption
Protein stabilization
mCherry
Two-dimensional nuclear magnetic resonance spectroscopy
Protein adsorption
Subjects
Details
- ISSN :
- 00063495
- Volume :
- 95
- Database :
- OpenAIRE
- Journal :
- Biophysical Journal
- Accession number :
- edsair.doi.dedup.....e6aad6ace28f8b24c4303f45d0311153