Osmolytes increase the stability of the folded form of protein and this effect has been exploited in pharmaceutical science, where these molecules have been used as protective "excipients" in high temperature and freezing steps. However, informations on the effects of osmolytes on the structure/dynamics of the native state, and therefore on the function of the macromolecules, are still scarce and controversial. In this work we have examined the effect of a model osmolyte, sucrose, in the dynamical structure of the native state of several modified azurins differing both in stability and internal flexibility. Protein dynamical structures have been investigated by Trp phosphorescence techniques, that in recent years have been proven to be able to reveal even subtle conformational changes. Changes in the flexibility of the proteins matrix were monitored by the intrinsic phosphorescence lifetime whereas more general effects on structural fluctuations were deduced from the acrylamide quenching rate constant, which measures the diffusion of the solute through the protein fold. We found that sucrose increases both the internal and the averaged rigidity of proteins, the effect is larger with the number of internal water molecules and the volume of free cavities. Moreover, the tightening effect becomes amplified at higher temperature, where the free volume inside the proteins is larger. The same results have been obtained with other sugars and polyols. For the proteins examined we found no relationship between the degree of stabilization and the increase in rigidity of the structure. This data supports the thesis that sucrose stabilizes proteins mainly by raising the free energy of the unfolded state. We conclude that, beside the effect of stabilization, osmolytes in solution reduce the flexibility of the polypeptides and, since protein dynamics is intimately associated with their function, they are likely to affect the activity of these macromolecules.
Effect of sucrose on the dynamics of the native state of proteins. A Trp phosphorescence study
Cioni P
2004
Abstract
Osmolytes increase the stability of the folded form of protein and this effect has been exploited in pharmaceutical science, where these molecules have been used as protective "excipients" in high temperature and freezing steps. However, informations on the effects of osmolytes on the structure/dynamics of the native state, and therefore on the function of the macromolecules, are still scarce and controversial. In this work we have examined the effect of a model osmolyte, sucrose, in the dynamical structure of the native state of several modified azurins differing both in stability and internal flexibility. Protein dynamical structures have been investigated by Trp phosphorescence techniques, that in recent years have been proven to be able to reveal even subtle conformational changes. Changes in the flexibility of the proteins matrix were monitored by the intrinsic phosphorescence lifetime whereas more general effects on structural fluctuations were deduced from the acrylamide quenching rate constant, which measures the diffusion of the solute through the protein fold. We found that sucrose increases both the internal and the averaged rigidity of proteins, the effect is larger with the number of internal water molecules and the volume of free cavities. Moreover, the tightening effect becomes amplified at higher temperature, where the free volume inside the proteins is larger. The same results have been obtained with other sugars and polyols. For the proteins examined we found no relationship between the degree of stabilization and the increase in rigidity of the structure. This data supports the thesis that sucrose stabilizes proteins mainly by raising the free energy of the unfolded state. We conclude that, beside the effect of stabilization, osmolytes in solution reduce the flexibility of the polypeptides and, since protein dynamics is intimately associated with their function, they are likely to affect the activity of these macromolecules.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
