Rubredoxin mutant A51C unfolding dynamics: A Förster Resonance Energy Transfer study

The unfolding dynamics of the rubredoxin mutant A51C (RdA51C) from Desulfovibrio vulgaris (DvRd) was studied on the temperature range from 25 °C to 90 °C and by incubation at 90 °C. By Förster Resonance Energy Transfer (FRET) the donor (D; Trp37) to acceptor (A; 1,5-IAEDANS) distance distribution was probed at several temperatures between 25 °C and 90 °C, and incubation times at 90 °C. From 25 °C to 50 °C the half-width distributions values (hw) are small and the presence of a discrete D-A distance was considered. At temperatures higher than 60 °C broader hw values were observed reflecting the existence of a distance distribution. The protein denaturation was only achieved by heating the solution for 2 h at 90 °C, as probed by the increase of the D-A mean distance. From Trp fluorescence it was shown that its vicinity was maintained until ∼ 70 °C, being the protein hydrodynamic radius invariant until 50 °C. However, at ∼ 70 °C a change in the partial unfolding kinetics indicates the disruption of specific H-bonds occurring in the hydrophobic core. The red shift of 13 nm, observed on the Trp37 emission, confirms the exposition of Trp to solvent after protein incubation at 90 °C for 2.5 h.