Probing the mechanism of rubredoxin thermal unfolding in the absence of salt bridges by temperature jump experiments.
Rubredoxins are the simplest type of iron-sulphur proteins and in recent years they have been used as model systems in protein folding and stability studies, especially the proteins from thermophilic sources. Here, we report our studies on the rubredoxin from the hyperthermophile Methanococcus jannaschii (T opt = 85 degrees C), which was investigated in respect to its thermal unfolding kinetics by temperature jump experiments. Different spectroscopic probes were used to monitor distinct structural protein features during the thermal transition: the integrity of the iron-sulphur centre was monitored by visible absorption spectroscopy, whereas tertiary structure was followed by intrinsic tryptophan fluorescence and exposure of protein hydrophobic patches was sensed by 1-anilinonaphthalene-8-sulphonate fluorescence. The studies were performed at acidic pH conditions in which any stabilising contributions from salt bridges are annulled due to protonation of protein side chain groups. In these conditions, M. jannaschii rubredoxin assumes a native-like, albeit more flexible and open conformation, as indicated by a red shift in the tryptophan emission maximum and 1-anilinonaphthalene-8-sulphonate binding. Temperature jumps were monitored by the three distinct techniques and showed that the protein undergoes thermal denaturation via a simple two step mechanism, as loss of tertiary structure, hydrophobic collapse, and disintegration of the iron-sulphur centre are concomitant processes. The proposed mechanism is framed with the multiphasic one proposed for Pyrococcus furiosus rubredoxin, showing that a common thermal unfolding mechanism is not observed between these two closely related thermophilic rubredoxins.
Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Bárbara J
BJ
Henriques
Lígia M
LM
Saraiva
Cláudio M
CM
Gomes
D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017017:Methanococcus / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D012416:Rubredoxins / Q000737:chemistry*; D012492:Salts; D013696:Temperature
D012416:Rubredoxins; D012492:Salts