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Upon addition of salt 2 M NaCl , the peak pattern in the spectrum expands B and the T 2 15 N of the main chain and the side chains can be discriminated inset of panel B , as expected for a folded protein of this size. The same trend is manifested in the peak intensities of the HSQC spectra and the traces for the considered peaks are also shown in the bottom right-hand corners of the spectra.
Solid lines in the figure insets correspond to the best exponential fitting to the experimental data filled circles. Surface residue replacements in the three proteins can increase or decrease the salt-induced stabilization or can even make it a destabilizing effect, depending on the nature and the number of surface residue replacements. Proteins with a decreased solvent accessible surface are preferred in an environment where water molecules also have to solvate the ions.
The stability of a halophilic protein at high salt concentrations will result from the balance of the side chain contributions to m salt and to the stability in the absence of cosolute. For the Kx n E mutation design in ProtL, chemical denaturation experiments revealed that the introduction of a. However, this is not the case for the majority of mutants studied in the present work where minor effects in stability have been found in the absence of salt, regardless of the nature or the number of substitutions. Examples of massive surface mutations into negative charged residues with minimal effects in stability have also been reported in the literature .
As shown in Figure 1 altering the number of negative charges on the protein surface results in almost no variation in stability modulation by salt indicating that D and N should be equally found in the aminoacid composition of halophilic proteins. D and E are among the hydrophilic residues that most contribute to increase protein solubility . Our solubility measurements with ProtL are consistent with this idea.
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By accumulating a large number of negative charged residues in the surface, the tendency to aggregation will be reduced and the solubility at physiological pH will increase due to the lowering in the isoelectric point. A decrease in the number of lysine residues results in an increase in halophilicity, independently of the charge, suggesting that the effect arises from the removal of the long lysine side chain.
Inspection of Figure 2 reveals that substitution of lysine by glutamate indeed causes an increase in and m salt white squares , consistent with the amino acid composition found in halophilic proteins. Moreover, structures of Kx5Q and Kx6E show that the replacement of lysine by glutamate results in a higher reduction of the solvent-exposed surface area than the substitution of glutamine. Depending on the natural environment, hypersaline adaptation involves KCl or NaCl.
However, our data are similar for both salts, indicating that a common mechanism prevails. The linear dependence of stability with the molar concentration of the salt suggests the existence of weak non-specific interactions  , . Some preliminary data with anions other than chloride indicate a cosolute-dependent modulation of protein stability following the Hofmeister series.
Thus, salt-induced stabilization can probably be described in terms of the preferential interaction and preferential exclusion of the ions from the protein surface.
The primers used to obtain the studied mutants are listed in the supplementary materials. Samples were grown in the appropriate media depending on the experiment: LB rich media for the CD, fluorescence, and calorimetric experiments and M9 minimal media with the appropriate isotope labeling for the NMR experiments. ProtL purification was achieved by a thermal shock followed by gel filtration chromatography Superdex 75, GE Healthcare and buffer exchange to 20 mM phosphate buffer at pH 6. For Ec 1ALigN, cell pellets were resuspended in 6 M guanidinium chloride and protein was refolded by fast dilution into 20 mM phosphate buffer, at pH 8.
The thermal range of the experiment was optimized for every sample, assuring the proper determination of the baselines for both the folded and the unfolded states. Between three and six independent measurements were obtained for each experimental condition from CD and fluorescence data, and duplicate points were used to obtain an estimation of the error. In all cases, the CD and fluorescence signal recovery after the thermal melt was monitored and is reported in the supplementary materials Tables S1 , S2 , S3.
A variation of T m with the scanning rate is an indication of irreversible unfolding and mutants showing such change within experimental error were excluded from the analysis red marks in Tables S1 , S2 , S3. Guanidinium chloride or urea denaturation experiments were followed by CD and used an initial volume of 1. Protein denaturation was achieved by the addition of aliquots of a solution at the same protein concentration but in 20 mM phosphate buffer and 6 M guanidinium chloride GuHCl or 10—11 M urea at pH 6. GuHCl and urea concentrations were adjusted by measuring the refractive index. A comparison of the unfolding free energies obtained by chemical denaturation with guadinium chloride and urea are shown in Table S5.
The free energy showed a linear dependence with the NaCl or KCl concentration, and the intrinsic stability in the absence of salt for this ProtL mutant subset and the stability at 3. The FF has been calculated from the expression: where and are the CD signal ellipticity at nm for the sample at the given KCl concentration and for the reference sample in 2M KCl , respectively. Data analysis was completed with in-house built scripts programmed in Matlab Simulink. The 20 conformers that represent the solution structure of the Kx5Q and Kx6E mutants have been deposited in the Protein Data Bank with the accession codes 2jzp and 2kac, respectively.
The ensemble constituted by the 10 lowest energy conformations from the solution NMR structures of wild type ProtL 2ptl  without the his-tag tail and the 10 lowest energy conformations for the Kx5Q ProtL 2jzp and Kx6E ProtL 2kac were employed for the solvent accessibility calculations, using the MOLMOL program  with a probe radius of 1. No experimental structural data are available for Q41, Q7, and E7. Very similar results were found for the areas determined from the experimental and the modeled structures, and the average areas for Q41, Q7, and E7 determined from the modeled structures were used in the analysis.
For Hv 1ALigN, the structural models succeeded in creating a structure from residues 33 to and this protein segment was used for the analysis of the including residues.
The 15 N side-chain transversal relaxation rates were measured on the NHD isotopmer to circumvent the contribution from proton dipole-dipole cross-correlation . Experimental data were adjusted to exponential decays to obtain the relaxation constant T 2 15 N.
The mixtures were allowed to equilibrate for at least 1 min and were then transferred to 1.
The concentrations of the supernatants were measured in a Nanodrop Thermo Scientific. Four independent solubility measurements were carried out for each protein. Experimental raw data. Salt concentration is colour coded following the legend shown in each panel. Fraction of protein folded versus the concentration of KCl.
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The error bars have been calculated from duplicate data. Buffer conditions: 20 mM phosphate buffer pH 8. Thermal denaturation T m values for ProtL.
Current Protein & Peptide Science | Bentham Science
Experimental mid denaturation points for the set of ProtL mutants as a function of the NaCl concentration. Error black bars represent the mean value for the duplicates. The lines represent the linear regressions for each specific dataset. Data for wild type ProtL are represented by blue circles and a blue line. All datasets are represented by filled symbols but the Kx n S mutants that are represented by open ones. Error bars represent the mean value for the duplicates. All datasets are represented by filled symbols but the Sx n K mutants are represented by open ones.
Correlation between the free energy of the mutants at 3. The expression: has been used to estimate the free energies at high salt concentration.
Values used for WT are: 4. Values used for the f factor are 3. Experimental m salt values for the cumulative mutants obtained from alternative mutation pathways. The alternative mutation pathways are shown in red. The protein target and the mutation class are specified in the enclosed legend.
Calculation of the m salt slopes from the experimental T m values. Primers for the ProtL multiple mutants and degree of reversibility upon thermal unfolding. Primers for the Hv 1ALigN multiple mutants and degree of reversibility upon thermal unfolding.
meble-grel.pl/wp-content Primers for the Ec 1ALigN multiple mutants and degree of reversibility upon thermal unfolding. We acknowledge Prof. Miquel Pons Barcelona University for helpful discussions and for constructive comments after reading the paper. The author s have made the following declarations about their contributions: Conceived and designed the experiments: OM. Wrote the paper: XT OM.
Abstract Proteins from halophilic organisms, which live in extreme saline conditions, have evolved to remain folded at very high ionic strengths. Author Summary Life on earth exhibits an enormous adaptive capacity and living organisms can be found even in extreme environments.