The Equilibrium Unfolding Intermediate Observed at pH 4 and its Relationship with the Kinetic Folding Intermediates in Green Fluorescent Protein

Enoki, Sawako; Maki, Kosuke; Inobe, Tomonao; Takahashi, Kazunobu; Kamagata, Kiyoto; Oroguchi, Tomotaka; Nakatani, Hiroaki; Tomoyori, Katsuaki; Kuwajima, Kunihiro

doi:10.1016/j.jmb.2006.07.009

Cited by 40 publications

(52 citation statements)

References 39 publications

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“…Folding trajectories probing equilibrium fluctuations and free energy profiles point to the presence of intermediates. We show, in agreement with experiments (33,38), using a variety of probes of the structures (distributions of the radius of gyration, R g , solvent accessible surface area, and structural overlap function, χ) that in the intermediate, the N-terminus is folded whereas the C-terminus region comprising of strands β 7 to β 10 are flexible and disordered. Upon initiating folding the time-dependent decrease in hR g ðtÞi, obtained using Brownian dynamics simulations (21,43), shows that compaction occurs in at least two major stages.…”

supporting

confidence: 87%

“…The increase in the contour length upon unfolding GFP from the folded to the intermediate state is consistent with formation of a structure in which strands (1-6) are intact and the rest are unfolded. Our simulations and previous ensemble experiments (33,37,38) have identified such an intermediate as occurring both in equilibrium and during the folding process.…”

Section: Discussionsupporting

confidence: 67%

“…However, the values of hR I g i and hR U g i obtained from simulations are considerably higher than the estimates from SAXS at pH ¼ 4 and pH ¼ 2.2, respectively. It is unclear if the greater compaction of I EQL and the unfolded state at acidic pH compared to simulations is due to the different conditions (pH versus temperature), the restricted range of scattering vectors (38) or due to the short comings of the SOP-SC model. The distribution PðR g Þ of R g shows that as [C] increases GFP samples extended conformations with R g exceeding 80° (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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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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supporting

confidence: 87%

Section: Discussionsupporting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Denaturant-dependent folding of GFP

Reddy

Liu

Thirumalai

2012

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

102

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“…Kuwajima and co-workers have studied the complex refolding kinetics of GFP from an acid-denatured state and proposed a model of folding in which the protein folds through parallel pathways and several intermediate states (Fukuda et al, 2000;Enoki et al, 2004). Recently, they have used small-angle x-ray scattering along with chromophore and tryptophan fluorescence studies to show that the two kinetic intermediates observed during refolding from pH 2.0 to 7.5 are closely related to an intermediate populated under equilibrium conditions at pH 4.0, which has molten-globule-like properties (Enoki et al, 2006). In previous studies, we have combined H/D exchange nuclear magnetic resonance (NMR) techniques with fluorescence measurements of chemically induced denaturation to establish that there is at least one stable and highly structured intermediate state populated under equilibrium conditions (Huang et al, 2007).…”

mentioning

confidence: 99%

The extremely slow‐exchanging core and acid‐denatured state of green fluorescent protein

et al. 2008

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Effects of IMAC specific peptide tags on the stability of recombinant green fluorescent protein

Widakowich

Zhang

Harris

et al. 2011

Biotechnology Progress

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Immobilized metal ion affinity chromatography (IMAC) using peptide affinity tags has become a popular tool for protein purification. An important feature dictating the use of a specific affinity tag is whether its structure influences the properties of the target protein to which it is attached. In this work we have studied the influence on protein stability of two novel peptide affinity tags, namely NT1A and HIT2, and compared their effect to the commonly used hexa-histidine tag, all attached to the C-terminus of a enhanced green fluorescent protein (eGFP). A comparison of the influence of C- or N-terminal orientation of the tags was also carried out by studying the NT1A tag attached at either terminus of the eGFP. Protein stability was studied utilising guanidine hydrochloride equilibrium unfolding procedures and CD and fluorescence spectroscopy. The novel peptide affinity tags, NT1A and HIT2, and the His6 tag were found to not affect the stability of eGFP. Although these results are protein specific, they highlight, nevertheless, the need to employ suitable characterisation tools if the impact of a specific peptide tag on the folded status or stability of a recombinant tagged protein, purified by immobilized metal ion affinity chromatographic methods, are to be rigorously evaluated and the appropriate choice of peptide tag made.

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The Equilibrium Unfolding Intermediate Observed at pH 4 and its Relationship with the Kinetic Folding Intermediates in Green Fluorescent Protein

Cited by 40 publications

References 39 publications

Denaturant-dependent folding of GFP

Denaturant-dependent folding of GFP

The extremely slow‐exchanging core and acid‐denatured state of green fluorescent protein

Effects of IMAC specific peptide tags on the stability of recombinant green fluorescent protein

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