Temperature and pressure dependence of protein stability: The engineered fluorescein-binding lipocalin FluA shows an elliptic phase diagram

Wiedersich, J.; Köhler, Simone; Skerra, Arne; Friedrich, J.

doi:10.1073/pnas.0710409105

Cited by 42 publications

(44 citation statements)

References 46 publications

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“…In conjunction with different molecular biology techniques such as direct mutagenesis, circular permutation and Förster resonance energy transfer, various fluorescent proteins have been developed to study the effects of intracellular properties including pH, Ca 2+ -concentration, and tensile force within a protein, on cellular behavior [6–9]. Despite their importance in numerous cellular processes, fluorescent proteins have not proven usable for measuring the physicochemical state of water, which affects protein functions, i.e., the enzymatic activity and structural stability of a protein strongly depend on the temperature and/or pressure of the solution [10–12]. Although the fluorescence intensity of fluorescent protein actually depends on the temperature and hydrostatic pressure, the intensity changes are too small to measure [13,14].…”

Section: Introductionmentioning

confidence: 99%

Glycine Insertion Makes Yellow Fluorescent Protein Sensitive to Hydrostatic Pressure

Watanabe

Imada

Yoshizawa³

et al. 2013

PLoS ONE

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Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP) by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the β-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure.

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Section: Introductionmentioning

confidence: 99%

Glycine Insertion Makes Yellow Fluorescent Protein Sensitive to Hydrostatic Pressure

Watanabe

Imada

Yoshizawa³

et al. 2013

PLoS ONE

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“…In addition, many globular proteins are also destabilized at low (subzero) temperatures, a process known as ''cold denaturation'' (8). Cold denaturation is experimentally accomplished with the help of elevated pressures (2), leading to a characteristic tongue-shaped P,T-stability diagram, found for many globular proteins (9)(10)(11)(12)(13)(14)(15). Hydrophobic forces play a key role in the protein folding process (16)(17)(18), but it is the balance of hydrophilic and hydrophobic forces that determines the conformational equilibrium.…”

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confidence: 99%

Computing the stability diagram of the Trp-cage miniprotein

Paschek

Hempel

Garcı́a

2008

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

150

194

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We report molecular dynamics simulations of the equilibrium folding/unfolding thermodynamics of an all-atom model of the Trp-cage miniprotein in explicit solvent. Simulations are used to sample the folding/unfolding free energy difference and its derivatives along 2 isochores. We model the ⌬G u(P,T) landscape using the simulation data and propose a stablility diagram model for Trp-cage. We find the proposed diagram to exhibit features similar to globular proteins with increasing hydrostatic pressure destabilizing the native fold. The observed energy differences ⌬E u are roughly linearly temperature-dependent and approach ⌬E u ‫؍‬ 0 with decreasing temperature, suggesting that the system approached the region of cold denaturation. In the low-temperature denatured state, the native helical secondary structure elements are largely preserved, whereas the protein conformation changes to an "open-clamp" configuration. A tighter packing of water around nonpolar sites, accompanied by an increasing solventaccessible surface area of the unfolded ensemble, seems to stabilize the unfolded state at elevated pressures.folding ͉ free energy ͉ hydrostatic pressure ͉ simulations T he stability of natively folded proteins in solution is determined by the competition of many effects that reach a balance near physiological conditions. As a consequence, the stability of a protein can be affected in many different ways (1-5). High-temperature denaturation is mostly accompanied by a dramatic loss of protein secondary structure (6). However, elevated pressures (2, 3), changing pH (2), and cosolvents such as salts (7) and osmolytes (4) also affect the stability of the native state, often destabilizing in character but under certain conditions also significantly stabilizing (4). In addition, many globular proteins are also destabilized at low (subzero) temperatures, a process known as ''cold denaturation'' (8). Cold denaturation is experimentally accomplished with the help of elevated pressures (2), leading to a characteristic tongue-shaped P,T-stability diagram, found for many globular proteins (9-15). Hydrophobic forces play a key role in the protein folding process (16-18), but it is the balance of hydrophilic and hydrophobic forces that determines the conformational equilibrium. The notion that proteins under native conditions are only ''marginally stable'' (7) seems to be an important requirement for their ability to explore different conformational substates (19) and hence for protein function. The application of high hydrostatic pressure (20) has been shown to be able shift the equilibrium of conformational states (21, 22), promoting denaturation (23) but also altering the native state (24) and modifying protein-protein interaction (20). Pressure effects on protein structure appear to be determined mostly by changing the balance between hydrophilic and hydrophobic interactions (25)(26)(27).Model peptides and proteins have long been sought as templates for understanding protein structure and function. The Trp-cage miniprotein (28) ...

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“…The binding of RecA protein from EC shows a hyperbolic phase diagram in the (P;T) plane near the transition to the commonly found elliptic phase diagrams found for the stability of various proteins (35,39,40). For TT, the region of the phase diagram above T > 60°C was not accessible, so we could not calculate the shape of the entire phase boundary like EC and extract thermodynamic parameters.…”

Section: Discussionmentioning

confidence: 90%

Effects of pressure and temperature on the binding of RecA protein to single-stranded DNA

Merrin

Kumar

Libchaber

2011

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

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The binding and polymerization of RecA protein to DNA is required for recombination, which is an essential function of life. We study the pressure and temperature dependence of RecA binding to single-stranded DNA in the presence of adenosine 5'-[γ-thio]triphosphate (ATP[γ-S]), in a temperature regulated high pressure cell using fluorescence anisotropy. Measurements were possible at temperatures between 5-60°C and pressures up to 300 MPa. Experiments were performed on Escherichia coli RecA and RecA from a thermophilic bacteria, Thermus thermophilus. For E. coli RecA at a given temperature, binding is a monotonically decreasing and reversible function of pressure. At atmospheric pressure, E. coli RecA binding decreases monotonically up to 42°C, where a sharp transition to the unbound state indicates irreversible heat inactivation. T. thermophilus showed no such transition within the temperature range of our apparatus. Furthermore, we find that binding occurs for a wider range of pressure and temperature for T. thermophilus compared to E. coli RecA, suggesting a correlation between thermophilicity and barophilicity. We use a two-state model of RecA binding/unbinding to extract the associated thermodynamic parameters. For E. coli, we find that the binding/ unbinding phase boundary is hyperbolic. Our results of the binding of RecA from E. coli and T. thermophilus show adaptation to pressure and temperature at the single protein level.pressure-temperature phase diagram | DNA binding protein | protein stability | protein functionality

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Temperature and pressure dependence of protein stability: The engineered fluorescein-binding lipocalin FluA shows an elliptic phase diagram

Cited by 42 publications

References 46 publications

Glycine Insertion Makes Yellow Fluorescent Protein Sensitive to Hydrostatic Pressure

Glycine Insertion Makes Yellow Fluorescent Protein Sensitive to Hydrostatic Pressure

Computing the stability diagram of the Trp-cage miniprotein

Effects of pressure and temperature on the binding of RecA protein to single-stranded DNA

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