Characterization of the Pressure-induced Intermediate and Unfolded State of Red-shifted Green Fluorescent Protein—A Static and Kinetic FTIR, UV/VIS and Fluorescence Spectroscopy Study

Herberhold, Heinz; Marchal, Stéphane; Lange, Reinhard; Scheyhing, Carsten H.; Vogel, Rudi F.; Winter, Roland

doi:10.1016/s0022-2836(03)00657-0

Cited by 100 publications

(81 citation statements)

References 51 publications

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“…Both its unfolding and refolding are slow and quite complex (8,11,12). The slowest observable phase in refolding of GFPuv occurs with a half-life of around 64 s at a GuHCl concentration of 0.06 M. This suggests high-energy barriers for both the unfolding and refolding reactions (48) (11)(12)(13)49). Using guanidine as the denaturant, we likewise found that the folding of GFPuv occurs in a stepwise manner and intermediates can be detected under both equilibrium and kinetic folding conditions ( Figures 2, 3 and 4; Tables 1 and 2).…”

Section: Discussionmentioning

confidence: 99%

Trigger Factor Assisted Folding of Green Fluorescent Protein

Xie

Zhou

2007

Biochemistry

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Guanidine induced equilibrium and kinetic folding of a variant of green fluorescent protein (F99S/M153T/V163A, GFPuv) was studied. Using manual mixing and stopped-flow techniques, we combined different probes, including tryptophan fluorescence, chromophore fluorescence and reactivity with DTNB, to trace the spontaneous and TF-assisted folding of guanidine denatured GFPuv. We found that both unfolding and refolding of GFPuv occurred in a stepwise manner and a stable intermediate was populated under equilibrium conditions. The thermodynamic parameters obtained show that the intermediate state of GFPuv is quite compact compared to the denatured state and most of the green fluorescence is retained in this state. By studying GFPuv folding assisted by TF and a number of TF mutants, we found that wild-type TF catalyzes proline isomerization and accelerates the folding rate at low TF concentrations, but retards GFPuv folding and decelerates the folding rate at high TF concentrations. This reflects the two activities of TF, as an enzyme and as a chaperone. A general mechanism of TF assisted protein folding is discussed.

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Section: Discussionmentioning

confidence: 99%

Trigger Factor Assisted Folding of Green Fluorescent Protein

Xie

Zhou

2007

Biochemistry

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“…Protoplasts of stationary phase cells were prepared as described by Weiss (17). One-milliliter samples (Laurdan embedded in E. coli protoplasts) were pressurized at 37°C by using a pressure cell with optical windows (7). Figure 1 shows the ␤-galactosidase activities resulting from HHP-dependent ToxR dimerization at 37°C.…”

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confidence: 99%

Influence of High Pressure on the Dimerization of ToxR, a Protein Involved in Bacterial Signal Transduction

Linke

Periasamy

Ehrmann

et al. 2008

Appl Environ Microbiol

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High hydrostatic pressure (HHP) is suggested to influence the structure and function of membranes and/or integrated proteins. We demonstrate for the first time HHP-induced dimer dissociation of membrane proteins in vivo with Vibrio cholerae ToxR variants in Escherichia coli reporter strains carrying ctx::lacZ fusions. Dimerization ceased at 20 to 50 MPa depending on the nature of the transmembrane segments rather than on changes in the ToxR lipid bilayer environment.

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“…Though developed at a light-gas gun facility, such shaped pressure-profiles are also feasible in principle with laser ablation or magnetic driven compression techniques allowing for new directions to be taken in high pressure physics. 62.50.+p; 1 The ability to attain extreme pressure and temperature conditions has given investigators in fields as diverse as biology, condensed matter physics, and earth and planetary sciences the tools to explore material behavior at megabar pressures and at thousands of degrees [1][2][3][4][5][6][7][8][9][10][11][12][13]. With these tools, they have simulated the physical conditions in the Earth's core and planetary interiors [1,2], probed a wide range of high pressure and temperature material properties [5][6][7][8][9][10], synthesized novel materials [11,12], and solved long standing physics problems such as the metallization of hydrogen [13].…”

Section: Introductionmentioning

confidence: 99%

High-pressure tailored compression: Controlled thermodynamic paths

Nguyen

Orlikowski

Streitz

et al. 2006

Journal of Applied Physics

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We have recently carried out novel and exploratory dynamic experiments where the sample follows a prescribed thermodynamic path. In typical dynamic compression experiments, the samples are thermodynamically limited to the principal Hugoniot or quasi-isentrope. With recent developments in the functionally graded material impactor, we can prescribe and shape the applied pressure profile with similarly-shaped, non-monotonic impedance profile in the impactor. Previously inaccessible thermodynamic states beyond the quasi-isentropes and Hugoniot can now be reached in dynamic experiments with these impactors. In the light gas-gun experiments on copper reported here, we recorded the particle velocities of the Cu-LiF interfaces and employed hydrodynamic simulations to relate them to the thermodynamic phase diagram. Peak pressures for these experiments were on the order of megabars, and the time-scales ranged from nanoseconds to several microseconds. The strain rates of the quasi-isentropic experiments are approximately 10 4 s −1 to 10 6 s −1 in samples with thicknesses up to 5 mm. Though developed at a light-gas gun facility, such shaped pressure-profiles are also feasible in principle with laser ablation or magnetic driven compression techniques allowing for new directions to be taken in high pressure physics. 62.50.+p; 1 The ability to attain extreme pressure and temperature conditions has given investigators in fields as diverse as biology, condensed matter physics, and earth and planetary sciences the tools to explore material behavior at megabar pressures and at thousands of degrees [1][2][3][4][5][6][7][8][9][10][11][12][13]. With these tools, they have simulated the physical conditions in the Earth's core and planetary interiors [1,2], probed a wide range of high pressure and temperature material properties [5][6][7][8][9][10], synthesized novel materials [11,12], and solved long standing physics problems such as the metallization of hydrogen [13]. These extreme conditions were achieved through three main techniques: static, shock and quasi-isentropic compressions [13][14][15][16][17][18][19][20][21][22][23][24]. Since the pioneering work by P. W. Bridgman [25], advances in diamond anvil cell technology have pushed the peak pressure by static compression from 200 kbars to more than 4 megabars [21,22]. Similarly, shock compression and quasi-isentropic compression techniques [13][14][15][16][17][18][19][20] can load samples to megabar pressures in a fraction of a nanosecond to microseconds.Future advances will likely push the peak pressure higher and extend the pressure loading time. However, these techniques will continue to be limited to a portion of the high pressure phase diagram by their characteristic loading rate and a single thermodynamic path. In particular, static compression yields continuous states on an isotherm (or an isochore, when heating), with a slow loading rate of˙ < 10 1 s −1 . Shock compression rapidly loads a sample at strain rates of˙ > ∼ 10 9 s −1 to a single state on the Hugoniot -...

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Characterization of the Pressure-induced Intermediate and Unfolded State of Red-shifted Green Fluorescent Protein—A Static and Kinetic FTIR, UV/VIS and Fluorescence Spectroscopy Study

Cited by 100 publications

References 51 publications

Trigger Factor Assisted Folding of Green Fluorescent Protein

Trigger Factor Assisted Folding of Green Fluorescent Protein

Influence of High Pressure on the Dimerization of ToxR, a Protein Involved in Bacterial Signal Transduction

High-pressure tailored compression: Controlled thermodynamic paths

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