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 degrees C to 90 degrees C and by incubation at 90 degrees 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 degrees C and 90 degrees C, and incubation times at 90 degrees C. From 25 degrees C to 50 degrees 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 degrees C broader hw values were observed reflecting the existence of a distance distribution. The protein denaturation was only achieved by heating the solution for 2h at 90 degrees C, as probed by the increase of the D-A mean distance. From Trp fluorescence it was shown that its vicinity was maintained until approximately 70 degrees C, being the protein hydrodynamic radius invariant until 50 degrees C. However, at approximately 70 degrees C a change in the partial unfolding kinetics indicates the disruption of specific H-bonds occurring in the hydrophobic core. The red shift of 13nm, observed on the Trp37 emission, confirms the exposition of Trp to solvent after protein incubation at 90 degrees C for 2.5h.
Santos
Andrea
A
Universidade de Lisboa, Av. Professor Egas Moniz, 1649-028 Lisboa, Portugal. andreasantos@fm.ul.pt
Duarte
Américo G
AG
Fedorov
Alexander
A
Martinho
José M G
JM
Moura
Isabel
I
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D016969:Desulfovibrio vulgaris; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D012416:Rubredoxins / Q000737:chemistry* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D001426:Bacterial Proteins; D050505:Mutant Proteins; D012416:Rubredoxins