The folding, stability and conformational dynamics of β-barrel fluorescent proteins

Hsu, Shang-Te Danny; Blaser, Georg; Jackson, Sophie

doi:10.1039/b908170b

Cited by 74 publications

(70 citation statements)

References 101 publications

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“…In recent years, much effort has been invested in understanding folding mechanisms and chromophore chemistry of naturally occurring fluorescent proteins GFP, RFP, and their derivatives (56). These proteins exhibit two distinct maturation phases, formation of a stable, 11-stranded ␤-barrel structure, followed by alignment, cyclization, and oxidation of a tripeptide chromophore within the shielded central cavity of the barrel.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of Ribosome-bound Fluorescent Proteins Reveals an Early, Stable, Cotranslational Folding Intermediate

Kelkar

Khushoo

Yang

et al. 2012

Journal of Biological Chemistry

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Background: Protein folding in cells is intimately coupled to protein synthesis. Results: GFP and RFP folding involves slow, cell autonomous insertion of the last ␤-strand into a stable cotranslational folding intermediate. Conclusion:Fluorescent ␤-barrel proteins utilize kinetically coupled co-and post-translational folding strategies. Significance: Specialized folding properties of GFP and RFP facilitate fluorescence acquisition in diverse biological environments.

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Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of Ribosome-bound Fluorescent Proteins Reveals an Early, Stable, Cotranslational Folding Intermediate

Kelkar

Khushoo

Yang

et al. 2012

Journal of Biological Chemistry

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“…Folding mechanisms of Green Fluorescent Protein (GFP), with predominantly β-sheet native structures stabilized by contacts between residues that are well separated along the sequence, have been extensively investigated (32)(33)(34)(35)(36)(37)(38)(39)(40). However, the details of the folding kinetics including the structures in the states that are visited along the folding routes are unexplored.…”

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confidence: 99%

“…1A), GFP has been used as a marker in a number of biotechnology and biophysical applications ranging from protein-protein interactions to gene expression (41). Consequently, it is important to map in detail the folding landscapes of FPs (32), which could aid in the development of new GFP tools for use in in vivo localization studies.…”

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confidence: 99%

Denaturant-dependent folding of GFP

Reddy

Liu

Thirumalai

2012

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

102

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We use molecular simulations using a coarse-grained model to map the folding landscape of Green Fluorescent Protein (GFP), which is extensively used as a marker in cell biology and biotechnology. Thermal and Guanidinium chloride (GdmCl) induced unfolding of a variant of GFP, without the chromophore, occurs in an apparent two-state manner. The calculated midpoint of the equilibrium folding in GdmCl, taken into account using the Molecular Transfer Model (MTM), is in excellent agreement with the experiments. The melting temperatures decrease linearly as the concentrations of GdmCl and urea are increased. The structural features of rarely populated equilibrium intermediates, visible only in free energy profiles projected along a few order parameters, are remarkably similar to those identified in a number of ensemble experiments in GFP with the chromophore. The excellent agreement between simulations and experiments show that the equilibrium intermediates are stabilized by the chromophore. Folding kinetics, upon temperature quench, show that GFP first collapses and populates an ensemble of compact structures. Despite the seeming simplicity of the equilibrium folding, flux to the native state flows through multiple channels and can be described by the kinetic partitioning mechanism. Detailed analysis of the folding trajectories show that both equilibrium and several kinetic intermediates, including misfolded structures, are sampled during folding. Interestingly, the intermediates characterized in the simulations coincide with those identified in single molecule pulling experiments. Our predictions, amenable to experimental tests, show that MTM is a practical way to simulate the effect of denaturants on the folding of large proteins.complex folding landscape of GFP | equilibrium and kinetic pathways | multiple folding routes | self-organized polymer model | multicanonical simulation O ur understanding of the folding mechanisms of small single domain proteins has advanced significantly over the last twenty years thanks to theoretical developments (1-7), simulations (8-13), and advances in experimental methods (14-19). Indeed, it can be justly stated that simulations of structure-based models are remarkably successful in predicting the folding mechanisms in the presence (20-22) and absence of denaturants (23-25). More refined questions, such as the relationship between pathway diversity and the symmetry of the underlying native structures (26), transition path times for crossing free energy barriers (27), the nature of the transition state ensemble (28-31) have also been addressed using theory and experiments. In contrast, much less is known about how large proteins with number, N, of amino acids exceeding ≈200 with complex β-sheet fold.Folding mechanisms of Green Fluorescent Protein (GFP), with predominantly β-sheet native structures stabilized by contacts between residues that are well separated along the sequence, have been extensively investigated (32-40). However, the details of the folding kinetics including the st...

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“…Similar to GFP, structural integrity of YFP is a requirement for its fluorescence property and the fluorescence is quenched upon denaturation. [70] Thus, it is evident that the immobilized YFP maintains its function on the hydrogel surface.…”

Section: Resultsmentioning

confidence: 99%

Photoinducedin situformation of clickable PEG hydrogels and their antibody conjugation

Kahveci

Çiftçi

Evran

et al. 2014

Designed Monomers and Polymers

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A simple approach for the preparation of a clickable poly(ethylene glycol)-based hydrogel as a polymeric support for protein immobilization via photoinitiated free radical polymerization of poly(ethylene glycol) diacrylate and propargyl acrylate is established. Bioconjugation to the obtained gels was achieved by azide-alkyne click reaction with azidofunctionalized anti-immunoglobulin G (anti-IgG) and anti-His tag antibodies. Evaluation of the affinities of the PEG hydrogels to the corresponding substrates (IgG and His-tagged YFP, respectively) indicates their specific binding capability.

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The folding, stability and conformational dynamics of β-barrel fluorescent proteins

Cited by 74 publications

References 101 publications

Kinetic Analysis of Ribosome-bound Fluorescent Proteins Reveals an Early, Stable, Cotranslational Folding Intermediate

Kinetic Analysis of Ribosome-bound Fluorescent Proteins Reveals an Early, Stable, Cotranslational Folding Intermediate

Denaturant-dependent folding of GFP

Photoinducedin situformation of clickable PEG hydrogels and their antibody conjugation

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