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Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.

ABSTRACT: Most thermophilic proteins tend to have more salt bridges, and achieve higher thermostability by up-shifting and broadening their protein stability curves. While the stabilizing effect of salt-bridge has been extensively studied, experimental data on how salt-bridge influences protein stability curves are scarce. Here, we used double mutant cycles to determine the temperature-dependency of the pair-wise interaction energy and the contribution of salt-bridges to ?C(p) in a thermophilic ribosomal protein L30e. Our results showed that the pair-wise interaction energies for the salt-bridges E6/R92 and E62/K46 were stabilizing and insensitive to temperature changes from 298 to 348 K. On the other hand, the pair-wise interaction energies between the control long-range ion-pair of E90/R92 were negligible. The ?C(p) of all single and double mutants were determined by Gibbs-Helmholtz and Kirchhoff analyses. We showed that the two stabilizing salt-bridges contributed to a reduction of ?C(p) by 0.8-1.0 kJ mol?¹ K?¹. Taken together, our results suggest that the extra salt-bridges found in thermophilic proteins enhance the thermostability of proteins by reducing ?C(p), leading to the up-shifting and broadening of the protein stability curves.

SUBMITTER: Chan CH

PROVIDER: S-EPMC3123365 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.

Chan Chi-Ho CH Yu Tsz-Ha TH Wong Kam-Bo KB

PloS one 20110624 6

Most thermophilic proteins tend to have more salt bridges, and achieve higher thermostability by up-shifting and broadening their protein stability curves. While the stabilizing effect of salt-bridge has been extensively studied, experimental data on how salt-bridge influences protein stability curves are scarce. Here, we used double mutant cycles to determine the temperature-dependency of the pair-wise interaction energy and the contribution of salt-bridges to ΔC(p) in a thermophilic ribosomal ...[more]

PMID: 21720566

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Project description:Enteroviruses are common agents of infectious disease that are spread by the fecal-oral route. They are readily inactivated by mild heat, which causes the viral capsid to disintegrate or undergo conformational change. While beneficial for the thermal treatment of food or water, this heat sensitivity poses challenges for the stability of enterovirus vaccines. The thermostability of an enterovirus can be modulated by the composition of the suspending matrix, though the effects of the matrix on virus stability are not understood. Here, we determined the thermostability of four enterovirus strains in solutions with various concentrations of NaCl and different pH values. The experimental findings were combined with molecular modeling of the protein interaction forces at the pentamer and the protomer interfaces of the viral capsids. While pH only had a modest effect on thermostability, increasing NaCl concentrations raised the breakpoint temperatures of all viruses tested by up to 20°C. This breakpoint shift could be explained by an enhancement of the van der Waals attraction forces at the two protein interfaces. In comparison, the (net repulsive) electrostatic interactions were less affected by NaCl. Depending on the interface considered, the breakpoint temperature shifted by 7.5 or 5.6°C per 100-kcal/(mol·Å) increase in protein interaction force.IMPORTANCE The genus Enterovirus encompasses important contaminants of water and food (e.g., coxsackieviruses), as well as viruses of acute public health concern (e.g., poliovirus). Depending on the properties of the surrounding matrix, enteroviruses exhibit different sensitivities to heat, which in turn influences their persistence in the environment, during food treatment, and during vaccine storage. Here, we determined the effect of NaCl and pH on the heat stability of different enteroviruses and related the observed effects to changes in protein interaction forces in the viral capsid. We demonstrate that NaCl renders enteroviruses thermotolerant and that this effect stems from an increase in van der Waals forces at different protein subunits in the viral capsid. This work sheds light on the mechanism by which salt enhances virus stability.

Project description:Halophilic proteins have greater abundance of acidic over basic and very low bulky hydrophobic residues. Classical electrostatic stabilization was suggested as the key determinant for halophilic adaptation of protein. However, contribution of specific electrostatic interactions (i.e. salt-bridges) to overall stability of halophilic proteins is yet to be understood. To understand this, we use Adaptive-Poison-Boltzmann-Solver Methods along with our home-built automation to workout net as well as associated component energy terms such as desolvation energy, bridge energy and background energy for 275 salt-bridges from 20 extremely halophilic proteins. We then perform extensive statistical analysis on general and energetic attributes on these salt-bridges. On average, 8 salt-bridges per 150 residues protein were observed which is almost twice than earlier report. Overall contributions of salt-bridges are -3.0 kcal mol-1. Majority (78%) of salt-bridges in our dataset are stable and conserved in nature. Although, average contributions of component energy terms are equal, their individual details vary greatly from one another indicating their sensitivity to local micro-environment. Notably, 35% of salt-bridges in our database are buried and stable. Greater desolvation penalty of these buried salt-bridges are counteracted by stable network salt-bridges apart from favorable equal contributions of bridge and background terms. Recruitment of extensive network salt-bridges (46%) with a net contribution of -5.0 kcal mol-1 per salt-bridge, seems to be a halophilic design wherein favorable average contribution of background term (-10 kcal mol-1) exceeds than that of bridge term (-7 kcal mol-1). Interiors of proteins from halophiles are seen to possess relatively higher abundance of charge and polar side chains than that of mesophiles which seems to be satisfied by cooperative network salt-bridges. Overall, our theoretical analyses provide insight into halophilic signature in its specific electrostatic interactions which we hope would help in protein engineering and bioinformatics studies.

Dataset Information

Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.

Publications

Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.

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