RESUMO
Thermodynamic stability parameters and the equilibrium unfolding mechanism of His 6HodC69S, a mutant of 1 H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) having a Cys to Ser exchange at position 69 and an N-terminal hexahistidine tag (His 6HodC69S), have been derived from isothermal unfolding studies using guanidine hydrochloride (GdnHCl) or urea as denaturants. The conformational changes were monitored by following changes in circular dichroism (CD), fluorescence, and dynamic light scattering (DLS), and the resulting transition curves were analyzed on the basis of a sequential three-state model N = I = D. The structural changes have been correlated to catalytic activity, and the contribution to stability of the disulfide bond between residues C37 and C184 in the native protein has been established. A prominent result of the present study is the finding that, independent of the method used for denaturing the protein, the unfolding mechanism always comprises three states which can be characterized by, within error limits, identical sets of thermodynamic parameters. Apparent deviations from three-state unfolding can be rationalized by the inability of a spectroscopic probe to discriminate clearly between native, intermediate, and unfolded ensembles. This was the case for the CD-monitored urea unfolding curve.
Assuntos
Arthrobacter/enzimologia , Dioxigenases/química , Dioxigenases/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Dobramento de Proteína , Ureia/farmacologia , Arthrobacter/efeitos dos fármacos , Arthrobacter/efeitos da radiação , Dicroísmo Circular , Dissulfetos/química , Luz , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Desnaturação Proteica/efeitos dos fármacos , Desnaturação Proteica/efeitos da radiação , Espalhamento de Radiação , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Stability, unfolding mechanism, spectroscopic, densimetric, and structural characteristics of the oxidatively stable C69S variant (HodC) of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) have been determined by classical and pressure modulation scanning calorimetry (DSC and PMDSC, respectively), circular dichroism (CD) spectroscopy, differential scanning densimetry (DSD), and dynamic light scattering measurements. At 25 degrees C, hexahistidine-tagged HodC has a hydrodynamic radius of 2.3 nm and is characterized by an unusually high degree of alpha-helical structure of approximately 60%, based on deconvolution of CD spectra. The percentage of beta-sheets and -turns is expected to be relatively low in view of its sequence similarity to proteins of the alpha/beta-hydrolase fold superfamily. His6HodC exhibits three-state unfolding (N <--> I <--> D) with an intermediate state I that exhibits at the transition temperature a volume larger than that of the native or denatured state. The intermediate state I is also associated with the highest isothermal expansion coefficient, alphaP, of the three states and exhibits a significantly lower percentage of alpha-helical structure than the native state. The stability difference between the native and intermediate state is rather small which makes I a potential candidate for reactions with various ligands, particularly those having a preference for the apparently preserved beta-type motifs.
Assuntos
Dioxigenases/química , Dobramento de Proteína , Sequência de Aminoácidos , Apoenzimas/química , Soluções Tampão , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Densitometria , Estabilidade Enzimática , Histidina/química , Histidina/isolamento & purificação , Temperatura Alta , Concentração de Íons de Hidrogênio , Luz , Modelos Químicos , Dados de Sequência Molecular , Peso Molecular , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espalhamento de Radiação , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
We report an extension of the recently published PMDSC method that permitted synchronous determination of heat capacity and expansibility when using slow, defined pressure formats in a DSC scan. Here we applied continuously opposing pressure changes that are fast compared to the time constants of the DSC instrument to study relaxation kinetics of phospholipids. Investigations of multilamellar vesicles of DPPC or DSPC in water revealed for both lipids relaxation times of about 30 s at the maximum of the main transition peak and about 15 s at the maximum of the pretransition. The relaxation times in the transition range are proportional to heat capacity of main- and pretransition. The molecular origin of the relaxation processes appears to stem from pressure-induced water fluxes between the interbilayer region and the bulk water phase.