RESUMEN
In order to elucidate the contribution of charged residues to protein stabilization at temperatures of over 100 °C, we constructed many mutants of the CutA1 protein ( EcCutA1) from Escherichia coli. The goal was to see if one can achieve the same stability as for a CutA1 from hyperthermophile Pyrococcus horikoshii that has the denaturation temperature near 150 °C. The hydrophobic mutant of EcCutA1 ( Ec0VV) with denaturation temperature ( Td) of 113.2 °C was used as a template for mutations. The highest Td of Ec0VV mutants substituted by a single charged residue was 118.4 °C. Multiple ion mutants were also constructed by combination of single mutants and found to have an increased thermostability. The highest stability of multiple mutants was a mutant substituted by nine charged residues that had a Td of 142.2 °C. To evaluate the energy of ion-ion interactions of mutant proteins, we used the structural ensemble obtained by a molecular dynamics simulation at 300 K. The Td of ionic mutants linearly increases with the increments of the computed energy of ion-ion interactions for ionic mutant proteins even up to the temperatures near 140 °C, suggesting that ion-ion interactions cumulatively contribute to the stabilization of a protein at high temperatures.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Iones/química , Proteínas Mutantes/química , Secuencia de Aminoácidos/genética , Estabilidad de Enzimas , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Calor , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Mutantes/genética , Conformación Proteica , TermodinámicaRESUMEN
Although the thermodynamics of protein denaturation at temperatures over 100 °C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (Td) ranging from 101 to 113 °C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mutant protein (Td = 137 °C) by substituting six residues with charged ones. Thermodynamic analyses of these mutant proteins indicated that the hydrophobic mutants were stabilized by the accumulation of denaturation enthalpy (ΔH) with no entropic gain from hydrophobic solvation around 100 °C, and that the stabilization due to salt bridges resulted from both the increase in ΔH from ion-ion interactions and the entropic effect of the electrostatic solvation over 113 °C. This is the first experimental evidence that has successfully overcome the typical technical difficulties.
Asunto(s)
Proteínas de Escherichia coli/química , Desnaturalización Proteica , Termodinámica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Calor , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Mutantes/química , Proteínas Mutantes/metabolismoRESUMEN
Unusually stable proteins are a disadvantage for the metabolic turnover of proteins in cells. The CutA1 proteins from Pyrococcus horikoshii and from Oryza sativa (OsCutA1) have unusually high denaturation temperatures (Td) of nearly 150 and 100 °C, respectively, at pH 7.0. It seemed that the CutA1 protein from the human brain (HsCutA1) also has a remarkably high stability. Therefore, the thermodynamic stabilities of HsCutA1 and its protease susceptibility were examined. The Td was remarkably high, being over 95 °C at pH 7.0. The unfolding Gibbs energy (ΔG(0)H2O) was 174 kJ/mol at 37 °C from the denaturant denaturation. The thermodynamic analysis showed that the unfolding enthalpy and entropy values of HsCutA1 were considerably lower than those of OsCutA1 with a similar stability to HsCutA1, which should be related to flexibility of the unstructured properties in both N- and C-terminals of HsCutA1. HsCutA1 was almost completely digested after 1-day incubation at 37 °C by subtilisin, although OsCutA1 was hardly digested at the same conditions. These results indicate that easily available fragmentation of HsCutA1 with remarkably high thermodynamic stability at the body temperature should be important for its protein catabolism in the human cells.
Asunto(s)
Proteínas de la Membrana/química , Secuencia de Aminoácidos , Encéfalo , Humanos , Datos de Secuencia Molecular , Replegamiento Proteico , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Desplegamiento Proteico , Proteolisis , Homología de Secuencia de Aminoácido , Subtilisina/química , TermodinámicaRESUMEN
To investigate the molecular basis of cold adaptation of enzymes, we determined the crystal structure of the tryptophan synthase α subunit (SfTSA) from the psychrophile Shewanella frigidimarina K14-2 by X-ray analysis at 2.6-Å resolution and also examined its physicochemical properties. SfTSA was found to have the following characteristics: (i) The stabilities against heat and denaturant of SfTSA were lower than those of an α subunit (EcTSA) from Escherichia coli. This lower equilibrium stability originated from both a faster unfolding rate and a slower refolding rate; (ii) the heat denaturation of SfTSA was completely reversible at pH 7.0 and the solubility of denatured SfTSA was higher than that of denatured EcTSA. The two-state transition of denaturation for SfTSA was highly cooperative, whereas the denaturation process of EcTSA was considerably more complex and (iii) the global structure of SfTSA was quite similar to those of α subunits from other species. Relative to those other proteins, SfTSA exhibited an increase in cavity volume and a decrease in the number of ion pairs. SfTSA also lacks a hydrogen bond near loop B, related to catalytic function. These characteristics of SfTSA might provide the conformational flexibility required for catalytic activity at low temperatures.
Asunto(s)
Adaptación Fisiológica , Frío , Shewanella/química , Shewanella/enzimología , Triptófano Sintasa/química , Triptófano Sintasa/metabolismo , Secuencia de Aminoácidos , Rastreo Diferencial de Calorimetría , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Conformación Molecular , Desnaturalización Proteica , Alineación de SecuenciaRESUMEN
Currently, there are no versatile and established methods for improving stability of proteins. In an entirely different approach from conventional techniques such as mutagenesis, we attempted to enhance enzyme stability of α-amylase from Aspergillus oryzae using a heavy-atom derivatization technique. We evaluated changes in stability using differential scanning calorimetry (DSC). Candidate heavy atoms were identified using the Heavy-Atom Database System HATODAS, a Web-based tool designed to assist in heavy-atom derivatization of proteins for X-ray crystallography. The denaturation temperature of α-amylase derivatized with gadolinium (Gd) or samarium (Sm) ions increased by 6.2 or 5.7°C, respectively, compared to that of the native protein (60.6°C). The binding of six Gd ions was confirmed by X-ray crystallography of the enzyme at 1.5 Å resolution. DSC and dynamic light-scattering data revealed a correlation between stability and the aggregation state upon addition of Gd ions. These results show that HATODAS search is an effective tool for selecting heavy atoms for stabilization of this protein.
Asunto(s)
Aspergillus oryzae/enzimología , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Gadolinio/farmacología , Samario/farmacología , alfa-Amilasas/química , alfa-Amilasas/metabolismo , Aspergillus oryzae/efectos de los fármacos , Rastreo Diferencial de Calorimetría/métodos , Cristalografía por Rayos X/métodos , Estabilidad de Enzimas , Calefacción , TermodinámicaRESUMEN
The CutA1 protein from Pyrococcus horikoshii (PhCutA1), a hyperthermophile, has an unusually high content of charged residues and an unusually high denaturation temperature. To elucidate the role of ion-ion interactions in protein stability, mutant proteins of PhCutA1 in which charged residues were substituted by noncharged residues were comprehensively examined. The denaturation temperatures (T(d)) for 13 of 53 examined mutant proteins were higher than that of the wild-type (148.5 °C at pH 7.0), among which E99Q had the highest T(d) at 154.9 °C. R25A had the largest decrease in T(d) among single mutants at ΔT(d) = -12.4 °C. The average decrease in T(d) of Lys or Arg mutants was greater than that of Glu or Asp mutants, and the average change in T(d) (ΔT(d)) of 21 Glu mutants was negligible, at 0.03 ± 2.05 °C. However, the electrostatic energy (-159.3 kJ·mol(-1)) of PhCutA1 was quite high, compared with that of CutA1 from Escherichia coli (-9.7 kJ·mol(-1)), a mesophile. These results indicate that: (a) many Glu and Asp residues of PhCutA1 should be essential for highly efficient interactions with positively charged residues and for generating high electrostatic energy, although they were forced to be partially repulsive to each other; (b) the changes in stability of mutant proteins with a T(d) value of ~140-150 °C were able to be explained by considering factors important for protein stability and the structural features of mutant sites; and (c) these findings are useful for the design of proteins that are stable at temperatures > 100 °C.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Ácido Aspártico/metabolismo , Ácido Glutámico/metabolismo , Estabilidad Proteica , Pyrococcus horikoshii/metabolismo , Sustitución de Aminoácidos , Proteínas Arqueales/genética , Ácido Aspártico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Glutámico/genética , Calor , Conformación Proteica , Desnaturalización Proteica , TermodinámicaRESUMEN
To enhance the heat stability of the CutA1 protein from Escherichia coli (EcCutA1) so that it has comparable stability to CutA1 from Pyrococcus horikoshii with a denaturation temperature (T(d)) of 150°C, we used the Stability Profile of Mutant Protein (SPMP) to examine the structure-sequence (3D-1D) compatibility between the conformation of EcCutA1 and its native sequence [J. Mol. Biol., 248, 733-738, (1995)]. We identified seven residues in EcCutA1 that were incompatible in terms of dihedral angles and hydrophobicity. These residues were replaced with appropriate amino acids, and the mutant proteins were evaluated for changes in stability by DSC and denaturant denaturation. The mutations that were introduced at five out of the seven positions improved the stability of EcCutA1. The T(d) values of single (S11A) and triple (S11V/E61V/Q73V) mutants improved by 16.5 and 26.6°C, respectively, compared to that of the wild-type protein (89.9°C). These analyses showed that (1) the stability of EcCutA1 is remarkably improved by slight substitutions, even though the stability of the wild-type protein is considerably high, (2) remarkable improvements in the stability can be quantitatively explained based on the newly solved native structure, and (3) SPMP is a powerful tool to examine substitutions that improve protein stability.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Calor , Estabilidad Proteica , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Desnaturalización Proteica , Ingeniería de Proteínas , Pyrococcus horikoshii/química , Pyrococcus horikoshii/genética , Relación Estructura-Actividad , TermodinámicaRESUMEN
The extended-spectrum beta-lactamases are associated with antibiotic resistance. Toho-1 R274N/R276N, a Class A beta-lactamase of CTX-M-type, efficiently hydrolyzes first generationcephalosporins (for example, cephalothin), in addition to cefotaxime, a third generation cephalosporin. However, this enzyme only marginally hydrolyzes the third generation cephalosporin ceftazidime, and the monobactam aztreonam. The deacylation defectiveness of the mutant Toho-1 E166A/R274N/R276N, which lacks the deacylation activity, results in the accumulation of the complex of an acylated-enzyme intermediate analog. For drug design, it would be useful if a quantitative prediction of a catalytic property were available without the need of enzymatic measurements. Therefore, we examined whether there is a correlation between the thermal stability of a catalytic intermediate (analog) and its kinetic parameters. First we measured the hydrolytic kinetics of the 14 species of beta-lactam antibiotics by Toho-1 R274N/R276N, and also measured the thermal stability of the accumulated acyl-intermediates of Toho-1 E166A/R274N/R276 by differential scanning calorimetry. Here we report the correlation of these parameters. The logarithm of the catalytic efficiency for Toho-1 R274N/R276N, log(k(cat)/K(m)) exhibited the best linear correlation with T(m,) which is the heat-denaturation temperature midpoint of the corresponding acylated complex of Toho-1 E166A/R274N/R276N. The correlation coefficient was 0.947, indicating that a relationship exists between the kinetic parameters and the stability of the intermediates. The results demonstrate a new method for investigating the catalytic properties of enzymes against any substrates, and a new approach to designing enzymes.