Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH

Abstract

Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT 2 and ReAsH-EDT 2 . This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide-biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.The physical phenomenon known as fluorescence has revolutionized cell biology, and its positive impact on molecular medicine will continue to develop with time. Driving this revolution is the green fluorescent protein (GFP) 1-3 and a rainbow of natural and engineered fluorescent protein (FP) variants that can be fused at the genetic level to proteins expressed in cell cultures and even whole animals 3 . Methodologies based on Förster resonance energy transfer (FRET) that use two or more FPs with overlapping absorption and emission spectra are used frequently as sensors to probe dynamic and complex processes including protein association, metal ion binding, conformational changes and post-translational modifications 4 . However, virtually all sensors based on FRET between FP color variants show dynamic changes in fluorescence intensity (typically 20-50%) that can be smaller than normal variations in cell-to-cell intensity due to differential FP expression 5 and that may be influenced significantly by Mg 2+ -ATP fluctuation 6 . Moreover, the steric bulk and slow folding of FPs limits their spatial and kinetic resolution 3,7 . We selected four structurally characterized polypeptides and protein domains to evaluate whether the Pro-Gly sequence within the linear tetracysteine motif could be replaced with one or more folded proteins while maintaining biarsenical affinity and fluorescence intensity. The feasibility of intramolecular bipartite tetracysteine display was evaluated using avian pancreatic polypeptide (aPP) 20 and Zip4 (ref. 21)-two well-folded polypeptides that were modified to contain one half of the linear tetracysteine motif at each terminus. Intermolecular bipartite tetracysteine display was evaluated using the protein-protein dimerization domains from the basic region leucine zipper (bZIP) proteins GCN4 (ref. 22) and Jun (ref. 23), which were modified to conta...

An uncoupling channel within the c-subunit ring of the F 1 F O ATP synthase is the mitochondrial permeability transition pore

“…To determine whether the c-subunit ring structure within the ATP synthase responds to CsA and Ca 2+ in cells, we adapted a fluorescent method, bipartite tetracysteine display (FlAsh), which detects protein-protein interactions in living cells and localizes proteins to subcellular compartments (24)(25)(26). When two pairs of cysteines placed on proteins come in close proximity, they bind tightly to the FlAsh dye, increasing its fluorescence intensity (Fig.…”

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

“…In this example, the FlAsH bound to a TC tag engineered into cellular retinoic acid-binding protein I had reduced fluorescence yield in the folded form relative to the unfolded form, and the folding could be tracked in E. coli cells. More recently protein folding and self-association was demonstrated in model proteins by bipartite tetracysteine display 6 . In this example, distal dicysteine pairs on model peptides were brought into close proximity upon binding of two peptides containing dicysteine pairs, or by the folding of a peptide that reconstitutes a functional tetracysteine motif for binding biarsenical dyes.…”

How to obtain labeled proteins and what to do with them

“…This method can be applied to study protein interactions with overexpressed proteins in the cell interior [54]. A number of examples have been published that utilize a split tetracysteine motif that can only be labeled if two proteins or protein domains interact [55][56][57]. This strategy has been applied to study protein association and protein folding.…”