RESUMO
The sperm whale apomyoglobin pH 4 folding intermediate exists in two forms, Ia and Ib, that mimic transient kinetic intermediates in the folding of the native protein at pH 6. To characterize the nature of the kinetic barrier that controls the formation of the earliest intermediate Ia, we have investigated the effects of small viscogenic cosolvents on its folding and unfolding kinetics. The kinetics are measurable by stopped-flow fluorescence and follow a cooperative two-state model in the absence and presence of cosolvents. Small cosolvents stabilize Ia, but, by applying the isostability test to separate the viscogenic effect of the cosolvent from its stabilizing effect, we found that, in both folding and unfolding conditions, the apparent rate constant decreases when solvent viscosity increases. The unitary inverse dependence of the apparent rate constant on solvent viscosity indicates a diffusion-controlled reaction. This result is consistent with the hypothesis that folding of the apomyoglobin pH 4 intermediate obeys a diffusion-collision model. Additionally, the temperature dependence of the reaction rate at constant viscosity indicates that the formation of Ia is also controlled by an energy barrier. Linear free energy relationships show that the transition state of the U <==> Ia reaction is compact and buries 45% of the surface area that is buried in native apomyoglobin. We conclude that the transition state of the U <==> Ia reaction resembles that for the formation of native proteins; namely, it is dry and its compactness is closer to that of the folded (Ia) form than of the unfolded form.
Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Mioglobina/química , Mioglobina/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Animais , Apoproteínas/isolamento & purificação , Dicroísmo Circular , Difusão , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Cinética , Movimento (Física) , Mioglobina/isolamento & purificação , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Solventes/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Cachalote , Sacarose/farmacologia , Temperatura , Termodinâmica , Ureia/farmacologia , ViscosidadeRESUMO
Several authors have reported that many sperm whale apomyoglobin mutants show anomalous circular dichroism spectra. These mutants have a low molar ellipticity compared to the wild-type protein but in several cases have the same stability of unfolding. A model in which native apomyoglobin is not folded in the same manner as that in other proteins and in which mutants show progressive reductions in their degree of folding has been suggested to explain this phenomenon. However, nuclear magnetic resonance of the native apomyoglobin conformation has shown that this state is folded and compact, raising the possibility that the anomalous circular dichroism spectra could have another explanation. We studied several mutants with anomalous circular dichroism spectra and found that these proteins were all contaminated with nucleic acid that contributed to the ultraviolet absorption and caused uncertainty in the determination of protein concentration. The resulting overestimation of the concentration of apomyoglobin explains the phenomenon of anomalous circular dichroism spectra. We describe a procedure to remove the contaminant nucleic acid which yields accurate protein concentration measurements and provides the normal circular dichroism spectra. Our findings support a well-structured native conformation for apomyoglobin and may also be of the interest to scientists working with the purification of recombinant proteins.
Assuntos
Apoproteínas/química , Dicroísmo Circular/métodos , Mioglobina/química , Animais , Apoproteínas/genética , Apoproteínas/isolamento & purificação , Concentração de Íons de Hidrogênio , Mutação , Mioglobina/genética , Mioglobina/isolamento & purificação , Baleias/metabolismoRESUMO
As molecular biology has developed it has become possible to abundantly produce heterologous proteins in bacteria and to design serial amino acid substitutions for the generation of modified proteins, an approach also known as protein engineering. Sperm whale myoglobin, a protein of broad interest, has been cloned for several years now and a large collection of mutants has been produced. The presence of heme stabilizes the protein, which is recovered soluble from the bacterial pellet, and most purification protocols take advantage of this property for myoglobin purification directly from the pellet. However, recovery from the column resin is poor with these methods making them expensive and the procedure for removing heme is laborious and drastic when the apo form of Mb is required. In the case of proteins with severe mutations, which bind heme weakly or do not bind it at all, such methods cannot be employed without massive loss of productivity. Here, we describe a modified method, which is both low cost and rapid, for the purification of the soluble apo form of Mb from Escherichia coli inclusion bodies. Biophysical characterization of the protein after purification shows that the purified apoMb retains its native conformation and is soluble. This modified method is also used for the purification of a non-heme-binding apoMb mutant, demonstrating its efficiency when dealing with drastic mutations.
Assuntos
Apoproteínas/isolamento & purificação , Heme/metabolismo , Mutação/genética , Mioglobina/genética , Mioglobina/isolamento & purificação , Sequência de Aminoácidos , Animais , Apoproteínas/química , Dados de Sequência Molecular , Mioglobina/química , Mioglobina/metabolismo , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de Tempo , BaleiasRESUMO
Se determinaron los valores de refernecia de apoproteínas A1 (apo A1) y B (apo B) para la población de Manizales. Para tal efecto fue realizado un muestreo estratificado proporcional de conglomerados. Sobre un total de 186 pacientes se realizaron cuantificaciones de lípidos séricos y apo A1 y apo B, y se calcularon los promedios poblacionales mediante un análisis de distribución 2 paramétrica log-normal. Sobre los datos obtenidos se calcularon los coeficientes de correlación (r), significancia estadística de tales coeficientes y análisis de regresión de los valores de aoproteínas con los lípidos séricos, edad y sexo. Los resulatdos obtenidos muestran diferencias en los niveles de apoproteínas por grupos de edad observándose valores inferiores de apo A1 y B en el grupo de personas de 15 a 24 años. los valores de apo A1 se incrementan hasta los 44 años en los hombres y van disminuyendo paulatinamente; en las mujeres el incremento se presenta hasta los 59 años y disminuye ligeramente después de los 60 años. Respecto a los valores de apo B se observa el mismo comportamiento. Sin embargo, la correlación apo A1 y B con edad es muy baja. Se presentaron diferencias estadísticamente significativas entre los promedios de apo A 1 y B por grupos de edad y según el sexo. Realizadas las correlaciones con los diferentes lípidos séricos los valores más altos observados fueron A1 con C-HDL (0.38) y de apo B con colesterol total (0.45), con C-LDL (0.34). La significancia de estas correlaciones es de P<0.001. Los valores de referencia obtenidos en este estudio son significativamente inferiores a los reportados en la literatura. Tales variaciones refuerzan la importancia de determinar valores de referencia que sean aplicables a la población objeto de atención en salud, dada la influencia de factores étnicos, ambientales, geográficos y nutricionales en los diferentes grupos poblacionales