RESUMO
Directed evolution techniques have been used to improve the thermal stability of the xylanase A from Bacillus subtilis (XylA). Two generations of random mutant libraries generated by error prone PCR coupled with a single generation of DNA shuffling produced a series of mutant proteins with increasing thermostability. The most Thermostable XylA variant from the third generation contained four mutations Q7H, G13R, S22P, and S179C that showed an increase in melting temperature of 20 degrees C. The thermodynamic properties of a representative subset of nine XylA variants showing a range of thermostabilities were measured by thermal denaturation as monitored by the change in the far ultraviolet circular dichroism signal. Analysis of the data from these thermostable variants demonstrated a correlation between the decrease in the heat capacity change (deltaC(p)) with an increase in the midpoint of the transition temperature (T(m)) on transition from the native to the unfolded state. This result could not be interpreted within the context of the changes in accessible surface area of the protein on transition from the native to unfolded states. Since all the mutations are located at the surface of the protein, these results suggest that an explanation of the decrease in deltaC(p) should include effects arising from the protein/solvent interface.
Assuntos
Bacillus subtilis/enzimologia , Evolução Molecular Direcionada , Endo-1,4-beta-Xilanases/química , Temperatura , Termodinâmica , Proteínas de Bactérias , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática/genética , Mutação de Sentido Incorreto , Desnaturação Proteica/genéticaRESUMO
Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca2+. However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.
Assuntos
Cardiomiopatias/genética , Tropomiosina/genética , Tropomiosina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Substituição de Aminoácidos , ATPase de Ca(2+) e Mg(2+)/antagonistas & inibidores , Cálcio/química , Cálcio/metabolismo , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Temperatura Alta , Humanos , Mutagênese Sítio-Dirigida , Concentração Osmolar , Pichia/genética , Pichia/metabolismo , Ligação Proteica , Desnaturação Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Termodinâmica , Tropomiosina/química , Tropomiosina/farmacologiaRESUMO
In triosephosphate isomerase, Cys126 is a conserved residue located close to the catalytic glutamate, Glu165. Although it has been mentioned that Cys126 and other nearby residues are required to maintain the active site geometry optimal for catalysis, no evidence supporting this idea has been reported to date. In this work, we studied the catalytic and stability properties of mutants C126A and C126S of Saccharomyces cerevisiae TIM (wtTIM). None of these amino acid replacements induced significant changes in the folding of wtTIM, as indicated by spectroscopic studies. C126S and C126A have K(M) and k(cat) values that are concomitantly reduced by only 4-fold and 1.5-fold, respectively, compared to those of wtTIM; in either case, however, the catalytic efficiency (k(cat)/K(M)) of the enzyme is barely affected. The affinity of mutated TIMs for the competitive inhibitor 2-phosphoglycolate augmented also slightly. In contrast, greater susceptibility to thermal denaturation resulted from mutation of Cys126, especially when it was changed to Ser. By using values of the rate constants for unfolding and refolding, we estimated that, at 25 degrees C, C126A and C126S are less stable than wtTIM by about 5.0 and 9.0 kcal mol(-)(1), respectively. Moreover, either of these mutations slows down the folding rate by a factor of 10 and decreases the recovery of the active enzyme after thermal unfolding. Thus, Cys126 is required for proper stability and efficient folding of TIM rather than for enzymatic catalysis.
Assuntos
Sequência Conservada , Cisteína/química , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/química , Triose-Fosfato Isomerase/química , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Catálise , Sequência Conservada/genética , Cisteína/genética , Ativação Enzimática/genética , Estabilidade Enzimática/genética , Glicolatos/química , Temperatura Alta , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Desnaturação Proteica/genética , Renaturação Proteica , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Triose-Fosfato Isomerase/biossíntese , Triose-Fosfato Isomerase/genéticaRESUMO
The effects of C-terminal truncation on the equilibrium folding transitions and folding kinetics of B. licheniformis exo small beta-lactamase (ES-betaL) have been measured. ES-betaL lacking 19 residues (ES-betaL(C)(Delta)(19)) has no enzymic activity. Deletion of the last 14 residues produces ES-betaL(C)(Delta)(14), which is 0.1% active. The enzyme lacking nine residues (ES-betaL(C)(Delta)(9)) is nearly fully active, has native optical and hydrodynamic properties, and is protease resistant, a distinguishing feature of the wild-type enzyme. Although ES-betaL(C)(Delta)(9) folds properly, it does so 4 orders of magnitude slower than ES-betaL, making possible the isolation and characterization of a compact intermediate state (I(P) ES-betaL(C)(Delta)(9)). Based on the analysis of folding rates and equilibrium constants, we propose that equilibrium between I(P) ES-betaL(C)(Delta)(9) and other intermediate slow folding. Residues removed in ES-betaL(C)(Delta)(9) and ES-betaL(C)(Delta)(14) are helical and firmly integrated into the enzyme body through many van der Waals interactions involving residues distant in sequence. The results suggest that the deleted residues play a key role in the folding process and also the existence of a modular organization of the protein matrix, at the subdomain level. The results are compared with other examples of this kind in the folding literature.