On the relative contribution of ionic interactions over iron-sulfur clusters to ferredoxin stability.
Metal centres play an important structural role in maintaining the native conformation of a protein. Here we use biophysical methods to investigate what is the relative contribution of iron-sulfur clusters in respect to ionic interactions in a thermophilic di-cluster ferredoxin model. Changes in protonation affect both the stability and the conformational dynamics of the protein fold. In the pH 5.5-8 interval, the protein has a high melting temperature (T(m) approximately 120 degrees C), which decreases towards pH extremes. Acidification triggers events in two steps: down to the isoelectric point (pH 3.5) the Fe-S clusters remain unchanged, the secondary structure content increases and the single Trp becomes more solvent shielded, denoting a more compact fold. Further acidification down to pH 2 sets off exposure of the hydrophobic core and Fe-S cluster disintegration, yielding a molten globule state. The relative stabilising contribution of the clusters becomes evident when stabilising ionic interactions are switched off as a result of poising the protein at pH 3.5, at an overall null charge: under these conditions, the Fe-S clusters disassemble at T(m)=72 degrees C, whereas the protein unfolds at T(m)=52 degrees C. Overall, this ferredoxin denotes a considerable structural plasticity around its native conformation, a property which appears to depend more on the integrity of its metal clusters rather than on the status of its stabilising electrostatic interactions. The latter however play a relevant role in determining the protein thermal stability.
Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Sónia S
SS
Leal
Cláudio M
CM
Gomes
D041541:Acidianus / Q000737:chemistry; D020816:Amino Acid Motifs / Q000502:physiology; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007477:Ions / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D055672:Static Electricity; D013455:Sulfur / Q000378:metabolism*; D013816:Thermodynamics
D005288:Ferredoxins; D007477:Ions; D013455:Sulfur; D007501:Iron