Temperature-induced conformational changes in turtle myoglobin

Abstract

A sea turtle myoglobin has been spin labelled in the cyanomet form (MbT-CN) with a maleimide spin label and temperature-induced changes were monitored by EPR. The EPR spectrum is composed by an immobilized and a more mobile signal. Comparison of this spectrum with spectra obtained from other proteins, blocking of the NH2-terminal and titration of SH groups lead to the conclusion that a reactive histidine is involved in the spin labelling. Our results on the temperature-induced changes monitored by the maleimide spin label suggest that a sequence of events is taking place in the transition which leads to protein denaturation (predenaturation): with increase in temperature atpH 8.5 the EPR parameter for the immobilized signal (α EPR) is the first to report the structural change (T 1/2=28°C); then the mobile signal changes (T 1/2=44°C) and finally the active centre monitored by optical absorption of the iron heme undergoes these changes (T 1/2=64°C). The activation energy calculated from the mobile spectrum is around 3 kcal/mol showing that these changes are quite subtle if compared to the enthalpy changes monitored by optical absorption of iron (40 kcal/mol). This predenaturation occurring in the range (30÷50)°C seems to be a general property of myoglobins, as has been observed also for whale and applysia.