Abstract
Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5–1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).
Highlights
Differential scanning calorimetry (DSC) is a powerful technique for studying thermal denaturation of globular proteins, and the methods for analysis of reversible thermal denaturation have been well elaborated [1,2,3,4]
It is strongly suggested that the irreversibly formed aggregates of MalZ have a definite size and a definite number of protein molecules, which remain preserved during the thermal denaturation. These results show that turbidity measurements together with DSC and circular dichroism (CD) measurements of the irreversible thermal denaturation of proteins provide a very effective approach for investigating the mechanisms of the irreversible denaturation of proteins
It should be noted that in the analysis of irreversible thermal transition by DSC, we focused on the kinetics represented by the Arrhenius equation (Eq (3)), and assumed that the DH was independent of T in a narrow temperature range of the thermal transition [48]
Summary
Differential scanning calorimetry (DSC) is a powerful technique for studying thermal denaturation of globular proteins, and the methods for analysis of reversible thermal denaturation have been well elaborated [1,2,3,4]. The thermal aggregation of globular proteins is usually not represented by a simple single-step process, but it may be assumed that the final aggregated entities are formed via starting assemblies [32,33,34,35,36,37]. The interrelationship between these processes and the irreversible thermal denaturation seems crucial for elucidating the molecular mechanisms of the thermal denaturation of proteins, but this interrelationship has been studied only for a very limited number of proteins [32,33,34,35,36,37]. The interrelationship between aggregation and denaturation may be important for understanding the mechanisms of human-disease-related amyloidogenesis of proteins, because the amyloidogenesis is brought about by partial denaturation followed by protein aggregation [13, 38]
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