In the present study the thermal unfolding of amicyanin, has been addressed using DSC, FE, OD, CD and EPR. The combined use of these techniques has allowed us to assess, during unfolding of the protein, its global conformational changes in relationship to the local structural modifications occurring in the copper environment and close to the fluorescent chromophore Trp 46 of the protein. The thermal transition from the native to the denatured state is on the whole irreversible and occurs in the temperature range between 65 and 72 °C depending on the scan rate and technique used. Amicyanin, as a whole, shows a complex unfolding pathway, which has been described in terms of a three-step model: N U F1 F2 According to this model, in the first step the native state of the protein, N, goes reversibly to the unfolded state U, in the second one U goes irreversibly to F1 and finally, the state F2 is irreversibly reached in the third step. Kinetic factors prevent the experimental separation of these steps. Nevertheless, the comparison of the data obtained with the different experimental techniques testifies the presence, within the unfolding pathway, of some intermediate states although not sufficiently long-lived to allow a detailed characterisation. A first intermediate transient state has been individuated around 68 °C, whereas a second one can be related to conformational changes that involve the copper environment. Finally, an exothermal phenomenon, caused by irreversible rearrangements of the melted polypeptide chains, is evidenced. In addition, according to the EPR findings, the type 1 copper ion, which is four fold co-ordinated by two N and two S atoms in a distorted tetrahedron in the native state of the protein, shows type 2 features after denaturation. A mathematical model simulating the unfolding Cpexc profile was also developed.
A model for the thermal unfolding of amicyanin / LA ROSA C.; MILARDI D.; GRASSO D.; VERBEET M. PH.; CANTERS G. W.; SPORTELLI L.; GUZZI R. - In: EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS. - ISSN 0175-7571. - 30(2002), pp. 559-570.
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|Titolo:||A model for the thermal unfolding of amicyanin|
|Data di pubblicazione:||2002|
|Citazione:||A model for the thermal unfolding of amicyanin / LA ROSA C.; MILARDI D.; GRASSO D.; VERBEET M. PH.; CANTERS G. W.; SPORTELLI L.; GUZZI R. - In: EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS. - ISSN 0175-7571. - 30(2002), pp. 559-570.|
|Appare nelle tipologie:||1.1 Articolo in rivista|