Researchers have engineered a blue fluorescent probe that is smaller than GFP and will improve the labeling of peptide-fused recombinant proteins in living cells. The group also developed a new protein labeling method, called probe incorporation mediated by enzymes (PRIME), which utilizes the new probes.
“Fluorescent proteins are the workhorses of fluorescent labeling methods and have been used to study proteins in living cells and in many biological applications,” Katharine White, a Massachusetts Institute of Technology (MIT) graduate student who participated in the research, told BioTechniques. “However, while perfectly specific, the large size of fluorescent proteins like GFP often disrupts the natural activity of proteins inside cells.”
While GFP is a protein that consists of 238 amino acids, the new technique uses a small, hydrophobic, and membrane-permeable fluorescent molecule. Once in the cell, and with the help of a modified enzyme, it binds to a transposable 13–amino acid peptide called LplA acceptor peptide (LAP2), which can be fused to a protein of interest.
White and colleagues, headed by MIT associate professor of chemistry Alice Ting, first engineered a fluorophore ligase derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structure-guided mutagenesis, LplA was modified so that instead of lipoic acid, its acceptor site recognizes the fluorophore 7-hydroxycoumarin.
Cells were then transfected with expression plasmids for the modified LplA enzyme and LAP2-fused protein of interest. After cells expressed both the LAP2 fusion proteins and LplA, they were incubated with the fluorophore. After washing, the localized 7-hydroxycoumarin–bound LAP2 proteins could be imaged.
The team used these much smaller probes with the PRIME method to effectively tag actin, a molecule involved in cell division, structure, motility, and communication. The relatively larger GFP interferes with the function and mobility of actin, but actin molecules tagged with the smaller PRIME probes were able to move throughout the cell and even enter the nucleus.
“The goal of creating the PRIME labeling method was to be able to study biology in living cells using a method that combined the small tag size of FlAsH with the high specificity and ease-of-use of GFP fusions,” said White.
According to White, PRIME is a better labeling method than even other peptide-based labeling systems. One such system, the FlAsH-EDT2 method developed by Roger Y. Tsien of the University of San Diego— who shared the 2008 Nobel Prize in chemistry with Osamu Shimomura and Martin Chalfie for the discovery of GFP—uses a small tag, but is plagued by specificity problems, which holds it back from replacing GFP.
“This method will provide a much needed alternative to GFP, and our ability to label subpopulations of a protein of interest is a unique and extremely powerful feature of PRIME labeling method,” said White. “The PRIME labeling method should provide life scientists with a way to label proteins of interest in a minimally invasive and extremely specific manner.”
Because the LAP2 peptide can be fused to a number of different proteins, the method enables the selective labeling of certain subpopulations of proteins in specific subcellular compartments. The researchers demonstrated PRIME’s specific subpopulation targeting by labeling proteins in only certain areas of a cell, including the nucleus, cell membrane, and cytosol.
The researchers intend to expand the technique to utilize different fluorescent probes and enzymes to capture the unique photo-switching capabilities of some fluorescent proteins. “One of the drawbacks of our current labeling method is that we are limited to ligation of a coumarin fluorophore,” said White. “What we would like to do is similarly expand our range of fluorophores that can be ligated using this methodology and evolve enzymes that can efficiently accept a variety of probes with different functional groups for applications such as single-molecule imaging and studying protein-protein interactions.”
Ting has filed for a patent on the PRIME technique and intends to commercialize the probes for use in other laboratories. The paper, “A fluorophore ligase for site-specific protein labeling inside living cells,” was published June 7 in Proceedings of the National Academy of Sciences.