A new twist on fluorescent tagging for proteins gives researchers a way to track folding and protein-protein interactions as they occur in living cells. The method, developed by Alanna Schepartz and colleagues at Yale University in New Haven, Connecticut, detects well-folded proteins using small probes that only fluoresce when they successfully bridge closely spaced pairs of cysteine residues. Changes in protein structure create visible differences in florescence signals in cells, the researchers show.
The paper, published in the November 4 online edition of Nature Chemical Biology, should interest AD researchers who are anxious for more tools to investigate the in-vivo behavior of amyloid-β (Aβ) peptides. Interest is high in figuring out how Aβ adopts the oligomeric conformations increasingly blamed for neurotoxicity (see ARF related news story). Indeed, Schepartz and colleagues write that the technique “may provide a means to detect early protein misfolding events associated with Alzheimer’s disease, Parkinson disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.”
The FlAsH and ReAsH tags Schepartz describes were first developed in Roger Tsien’s lab at the University of California, San Diego. The compounds are derivatives of fluorescein and resorufin, coupled with two reactive arsenic groups to produce cell-permeable, non-fluorescent compounds. When the compounds react in a sequence-specific way with the cysteine residues of a CCPGCC tag engineered into proteins, their fluorescence is restored and the result is a protein bearing a covalently linked fluorescent chromophore.
The tagging reaction depends on the close proximity of the cysteine pairs, which must be within about 7 angstroms for labeling to occur. That led the Yale researchers to ask if the tags would label cysteines that were brought together by protein folding or by protein-protein interactions, rather than sitting adjacent to each other in the peptide sequence. First author Nathan Luedtke tested this idea using model proteins with well-characterized structures, either monomeric polypeptides or a pair that dimerizes via a leucine zipper motif. For the monomers, Luedtke used proteins whose proper folding brought the amino and carboxy terminals into close association, and placed half the cysteine tag on each end. The dimerizing proteins each got half of the cysteine tag.
The technique worked like a charm, and in vitro measurements revealed that proteins with the split cysteine sequence showed an affinity for labeling and a brightness that was comparable to proteins with an intact CCPGCC sequence. The labeling depended on proper folding or protein-protein association, as point mutations that disrupted structure also decreased protein fluorescence by two- to 10-fold.
The same dependence on proper folding was also seen for in vivo labeling. In cells transfected with tagged proteins and then exposed to ReAsH, the investigators saw bright fluorescence with wild-type proteins, but only weak signals from the mutated polypeptides.
The new technique offers several advantages over existing fluorescence labeling methods, the authors write. The FlAsH and ReAsH groups are small, so may have less chance of interfering with protein function than larger fusion partners such as green fluorescent protein. The FlAsH/ReAsH method offers better spatial resolution than fluorescence resonance energy transfer (FRET), a commonly used measure of proximity that at best detects groups 20 angstroms or less apart. FRET uses multicolor imaging, while with FlAsH/ReAsH, the differences in fluorescence in cells expressing folded versus unfolded proteins could be made out by eye under the microscope. The sensitivity and simplicity may make the technique suitable for high-throughput screening for compounds that stabilize particular protein folds, or for early detection of protein misfolding in AD or PD.—Pat McCaffrey