Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

Abstract

In vivo fluorescent labeling of an expressed protein has enabled the observation of its stability and aggregation directly in bacterial cells. Mammalian cellular retinoic acid-binding protein I (CRABP I) was mutated to incorporate in a surface-exposed omega loop the sequence Cys-Cys-Gly-Pro-Cys-Cys, which binds specifically to a biarsenical fluorescein dye (FlAsH). Unfolding of labeled tetra-Cys CRABP I is accompanied by enhancement of FlAsH fluorescence, which made it possible to determine the free energy of unfolding of this protein by urea titration in cells and to follow in real time the formation of inclusion bodies by a slow-folding, aggregationprone mutant (FlAsH-labeled P39A tetra-Cys CRABP I). Aggregation in vivo displayed a concentration-dependent apparent lag time similar to observations of protein aggregation in purified in vitro model systems.protein aggregation ͉ protein folding ͉ fluorescence P rotein folding is an exquisitely optimized process that normally succeeds in producing functional molecules in vivo, despite physicochemical conditions that are very challenging. The heteropolymeric nature of the polypeptide chain encodes a great diversity of complex architectures of native proteins but also presents many side chain groups that are marginally soluble in aqueous solution. Not surprisingly, aggregation of newly synthesized proteins emerges as a process that competes with folding in vivo. In bacterial cells, aggregation of partially folded intermediates manifests itself in the production of insoluble inclusion bodies and often occurs when an exogenous protein is overexpressed (1). Several human diseases, including Alzheimer's, Huntington's, and spongiform encephalopathies, are characterized by the formation of insoluble protein aggregates (2). These distinctive aggregated states seem to be formed from partially folded, partially unfolded, or other nonnative intermediates and not from the native state. Thus, considerable research has been dedicated to the goal of achieving a fundamental understanding of protein aggregation, and much progress has been achieved in several systems in vitro (3). By contrast, relatively little is known about how this process occurs in vivo. Complicating research on protein folding and aggregation in vivo is the need to study these processes in the complex and concentrated environment of the cell. It is therefore highly desirable to develop methods to observe the fates of proteins directly in cells, including their thermodynamic stabilities, their kinetics of folding, the nature of folding intermediates and the energy landscape of folding, and the effects of mutations. Here, we have taken advantage of new approaches to labeling protein molecules in the cell with fluorescent dyes (4) to monitor the synthesis, folding, and aggregation of a protein whose in vitro folding has been well characterized.Cellular retinoic acid-binding protein I (CRABP I) is a 136-residue member of the large family of intracellular lipidbinding proteins, which fold into ␤-barrel structures (Fi...

Soft interactions and crowding

“…Their main conclusion was that stability is unchanged in cells. The Gierasch laboratory fluorescently tagged cellular retinoic acid-binding protein I (16 kDA) in cells and measured stability by using urea denaturation (Ignatova and Gierasch 2004;Ignatova et al 2007). They showed that the test protein was destabilized in cells.…”

“…(We refer readers to their chapter in this volume for details.). They utilized the model to simulate the systems described above (Ghaemmaghami and Oas 2001;Ignatova and Gierasch 2004) using four scenarios. The first scenario considered only hard-core repulsions and, as predicted, the result was stabilization.…”

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

“…The Ω-loop shows the lowest sequence conservation among the intracellular lipid-binding protein family (Gunasekaran et al, 2004) and tolerates sequence expansions and deletions (Ignatova and Gierasch, 2004). By this intramolecular insertion of the FlAsH-binding motif, we found that the FlAsH-fluorescence emission signal reports on the conformational state of the protein, with misfolded, denatured, and aggregated states hyperfluorescent compared to the native state (Ignatova and Gierasch, 2004). This system allowed us to measure in vivo stability using a urea titration (Ignatova and Gierasch, 2004;Ignatova et al, 2007a).…”

“…Here we describe a fluorescence-based approach that we have recently developed to determine protein stability in vivo (in Escherichia coli cells) and in vitro (Ignatova and Gierasch, 2004) and that may be used to monitor in vivo folding propensities and aggregation kinetics in real time and in a noninvasive manner. The protein of interest is visualized in the context of all cellular macromolecules using the membrane-permeable bis-arsenical fluorescein-based dye "FlAsH" (named originally as a "fluorescein arsenical helix" binder), which specifically ligates to a genetically engineered, highly uncommon tetracysteine motif (Cys-Cys-Xxx-Yyy-CysCys) (Griffin et al, 2000).…”

“…We integrated the tetracysteine sequence (here Cys-Cys-Gly-Pro-Cys-Cys, incorporating the native Gly-Pro present in this loop) into an internal Ω-loop of the 136-amino acid cellular retinoic acid-binding protein I (referred to here as CRABP) (Ignatova and Gierasch, 2004). The Ω-loop shows the lowest sequence conservation among the intracellular lipid-binding protein family (Gunasekaran et al, 2004) and tolerates sequence expansions and deletions (Ignatova and Gierasch, 2004).…”