Combined spectroscopic and calorimetric characterisation of rubredoxin reversible thermal transition.
Rubredoxins are small iron proteins containing the simplest type of iron-sulphur centre, consisting of an iron atom coordinated by the thiol groups of four cysteines. Here we report studies on the conformational stability of a new type of rubredoxin from the hyperthermophile Methanocaldococcus jannaschii, having an atypical metal site geometry resulting from a modified iron-binding motif. Absorption and fluorescence spectroscopies were used in combination with differential scanning calorimetry to probe different aspects of the thermal unfolding transition: iron site degradation (absorption at 380 nm), tertiary structure unfolding (Trp emission), exposure of hydrophobic regions (1-anilinonaphalene-8-sulphonate fluorescence enhancement) and iron release. Thermal denaturation was found to be irreversible and caused by decomposition of the metal centre. The protein is hyperstable and between pH 4 and 10 it is only thermally denatured in the presence of a strong chemical denaturant. The study of the heating rate dependence of the melting temperature allowed us to determine the reaction equilibrium thermodynamic parameters. At pH 2 the protein is destabilised owing to the absence of salt bridges and it has a T(m) of 65 degrees C. In these conditions, there is excellent agreement between the parameters determined by the different spectroscopic methods and calorimetry. The highest stability was found to be at pH 8, and a detailed study of the heating rate dependence in the presence of guanidine thiocyanate in this condition allowed the determination of a reversible T(m) of 118 degrees C.
Henriques
Bárbara J
BJ
Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. República 127, 2780-756 Oeiras, Portugal.
Saraiva
Lígia M
LM
Gomes
Cláudio M
CM
D001665:Binding Sites; D002152:Calorimetry, Differential Scanning / Q000379:methods; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007501:Iron / Q000737:chemistry*; D017017:Methanococcus / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012416:Rubredoxins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics
D012416:Rubredoxins; D007501:Iron