title: Quantitative fluorescence correlation spectroscopy reveals a 1000-fold increase in lifetime of protein functionality.
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 23 muM 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.
authors.affiliation: MESA+ Institute for Nanotechnology, Biophysical Engineering Group, University of Twente, Enschede, The Netherlands.
authors.forename: Dianwen
authors.initials: D
authors.lastname: Zhang
authors.forename: Hannes
authors.initials: H
authors.lastname: Lans
authors.forename: Wim
authors.initials: W
authors.lastname: Vermeulen
authors.forename: Aufried
authors.initials: A
authors.lastname: Lenferink
authors.forename: Cees
authors.initials: C
authors.lastname: Otto
mesh_terms: D000327:Adsorption; D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013499:Surface Properties; D013501:Surface-Active Agents / Q000494:pharmacology; D013997:Time Factors; D014867:Water / Q000737:chemistry
chemical_list: D050505:Mutant Proteins; D011506:Proteins; D013501:Surface-Active Agents; D014867:Water; D004247:DNA; D004262:DNA Restriction Enzymes