FRET-based in Vivo Screening for Protein Folding and Increased Protein Stability

Philipps, Björn; Hennecke, Jens; Glockshuber, Rudi

doi:10.1016/s0022-2836(03)00077-9

Cited by 48 publications

(36 citation statements)

References 34 publications

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“…Then, if BFP and GFP are in sufficiently close proximity, efficient FRET from BFP to GFP is observed in vivo in E. coli cells, leading to green fluorescence. The theoretically predicted R o value (i.e., the distance between donor and acceptor where FRET efficiency is half-maximal) for the BFP-GFP donor-acceptor couple is 41 Å (28). The distance between the buried chromophores and the surface of the fluorescent proteins amounts to Ϸ8 Å (28).…”

Section: Resultsmentioning

confidence: 99%

Membrane association of myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain and a coiled-coil dimerization module

Berger¹,

Schaffitzel²,

Berger³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

105

128

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Mutations in the myotubularin (MTM)-related protein 2 (MTMR2) gene are responsible for the severe autosomal recessive neuropathy Charcot-Marie-Tooth disease type 4B1. MTMR2 belongs to the MTM family of dual-specific phosphatases that use phosphatidylinositol (PI) 3,5-bisphosphate [PI(3,5)P 2] and PI 3-phosphate [PI(3)P] as their substrate. Because these substrates are localized in the membrane bilayer, membrane targeting of Mtmr2 is an important regulatory mechanism. In hypoosmotically stressed COS cells with increased levels of PI(3,5)P 2, Mtmr2 is bound to the membrane of vacuoles formed under these conditions. Using several mutant forms of Mtmr2, we identified two domains that are necessary for membrane association: (i) A pleckstrin homology-GRAM domain; and (ii) a coiled-coil module. Protein-lipid overlay assays show that the pleckstrin homology-GRAM domain binds to PI(3,5)P 2 and PI(5)P, a substrate and a product of the Mtmr2 enzyme, respectively. We also demonstrate that Mtmr2 forms a dimer and that the C-terminal coiled-coil is responsible for homodimerization, in addition to membrane association. Our data indicate that phosphoinositide-protein interactions, as well as protein-protein interactions, are necessary for the correct regulation of MTMR2.

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Section: Resultsmentioning

confidence: 99%

Membrane association of myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain and a coiled-coil dimerization module

Berger¹,

Schaffitzel²,

Berger³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

105

128

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“…However, studies that allow direct observation of aggregation without analysis of cell constituents after lysis are rare (31,32 (33) designed an in vivo protein folding assay using N-terminal fusions to GFP to identify proteins that did not aggregate, but did not directly observe aggregation events. Philipps et al (34) proposed a screening approach to observe folding and stability based on fluorescence resonance energy transfer between flanking C-terminal GFP and N-terminal blue fluorescent protein.…”

Section: Discussionmentioning

confidence: 99%

Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

Ignatova

Gierasch

2003

Proc. Natl. Acad. Sci. U.S.A.

227

239

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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...

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“…[4][5][6] The effects of mutations on thermodynamic stability in vitro are well documented. 7 Although several elegant studies revealed aspects of protein stability in vivo, [8][9][10][11] we still do not fully understand how the reversible unfolding of proteins in the testtube relates to the environment of the cell. There are Present address: S. Rüdiger, Cellular Protein Chemistry, Utrecht-University, Padualaan 8, 3584 CH Utrecht, The Netherlands.…”

Section: Introductionmentioning

confidence: 99%

Correlation of Levels of Folded Recombinant p53 in Escherichia coli with Thermodynamic Stability in Vitro

Mayer

Rüdiger

Ang

et al. 2007

Journal of Molecular Biology

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The amount of folded functional protein in a cell is controlled by a number of factors, including the relative rates of its biosynthetic and specific degradation processes, and its intrinsic thermodynamic stability. Mutationinduced loss of stability is a common cause of disease. Many oncogenic mutants of the tumour suppressor p53, for example, reduce the intrinsic thermodynamic stability of the protein in vitro. We have analysed the level of recombinant folded human p53 core domain (p53C) and its mutants in Escherichia coli spanning a stability range of 6 kcal/mol to assess the effects of intrinsic thermodynamic stability in vivo in the absence of specific ubiquitin-mediated pathways in human cells. The levels of folded protein were measured fluorimetrically in living cells by fusing the gene of p53C upstream to that of green fluorescent protein and measuring the fluorescence relative to a control at various temperatures. At a fixed temperature, the amount of fluorescence is correlated with the thermodynamic stability of the mutant. The level of each protein varied with temperature according to a sigmoid curve that paralleled the melting in vitro, but the apparent T m was lower in vivo, because steady-state levels are observed rather than true thermodynamic equilibria. Our results show clearly that changes in the intrinsic thermodynamic stability of p53 reduce the level of folded and hence functional p53 substantially in E. coli, and provide insights into the correlation between protein instability and disease at the cellular level.

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FRET-based in Vivo Screening for Protein Folding and Increased Protein Stability

Cited by 48 publications

References 34 publications

Membrane association of myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain and a coiled-coil dimerization module

Membrane association of myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain and a coiled-coil dimerization module

Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

Correlation of Levels of Folded Recombinant p53 in Escherichia coli with Thermodynamic Stability in Vitro

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