Chapter 3 A Fluorescent Window Into Protein Folding and Aggregation in Cells

Abstract

Evolutionary selective pressures have tuned the efficiency of the protein-folding reaction in the crowded complex environment in the cell. Nevertheless, the fidelity of folding is imperfect, leading to off-pathway intermolecular interactions that compete with proper folding and to consequent formation of thermodynamically stable aggregates. Such aggregates constitute the histopathological hallmarks of many neurodegenerative pathologies. Yet, most of the approaches to characterize protein folding and/or misfolding are limited to in vitro conditions. Here, we describe a strategy to directly monitor the behavior of a protein in prokaryotic and eukaryotic cells. The method is based on incorporation of structurally non-perturbing, specific binding motifs for a bis-arsenical fluoroscein dye, FlAsH, in sites that result in distinct dye fluorescence signals for the folded and unfolded states of the protein under study. Our approach has been developed using as a case study the predominantly β-sheet intracellular lipid-binding protein, cellular retinoic acid-binding protein, alone or as a chimera fused to the exon 1-encoded fragment of huntingtin, which harbors a polyglutamine repeat tract. We have designed protocols to label this protein in vivo and to monitor the resulting fluorescence signal, which reports on any misfolding transition and formation of aggregates, yielding quantitatively interpretable data.

Coumarin 6 and 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent probes to monitor protein aggregation

“…2 Recently, imaging techniques using a bisarsenoid fluorescein analogue dye, viz. FlAsH, that is able to distinguish between folded and unfolded states of proteins without interfering with the protein structure 3 and JC-1 that is able to distinguish between monomeric and fibrillar forms of proteins, 4 have been reported. However, fluorescence microscopy cannot be the method of choice when libraries of compounds need to be screened as modulators of protein aggregation.…”

Protein Fibrillation under Crowded Conditions

“…For more than 40 years, scientists have characterized the effects of the cellular interior on protein function. The advances in technology, such as in-cell NMR [ 9 , 99 ], single-cell mass spectrometry [ 100 ], fluorescence [ 8 , 10 , 101 , 102 ], FRET [ 103 ] and flow cytometry [ 104 ], electron microscopy [ 5 , 6 , 7 ], and cryo-electron tomography (Cryo-ET), allow scientists to characterize the protein and the protein aggregate structure and function in cells. Cryo-ET is particularly effective at structural characterization of the neurotoxic aggregates [ 105 , 106 , 107 ].…”

“…The work of the Bhat group has recently sought to understand the effect of polyol osmolytes on protein fibrillation, specifically, how the addition of -OH groups alters fibrillation [88,89]. The molecules utilized are ethylene glycol (2 -OH groups), glycerol (3), erythritol (4), xylitol (5), and sorbitol (6). The work of Roy and Bhat dug deeper into the effects of osmolytes in human γ-synuclein which, in the same way as α-synuclein, is intrinsically disordered [88].…”

“…In the cells and extracellular matrices, the proteins fold and misfold in a crowded environment, surrounded by a complex (and nonrandom) mixture of other solutes [ 3 , 4 ]. Ideally, fibrillation would primarily be studied in living organisms [ 5 , 6 , 7 , 8 , 9 , 10 ], however, measuring the kinetics of fibrillation in the cells poses obvious technical challenges. Instead, researchers have attempted to mimic the crowded environment of cells in vitro, via crowding agents [ 11 ].…”

Why and how protein aggregation has to be studied in vivo

“…This smart approach enables to monitor the formation of hyperfluorescent aggregates within intact cells, by simply detecting the bulk cell fluorescence or by fluorescence microscopy [30,31]. …”