Structural stabilization of [2Fe-2S] ferredoxin from Halobacterium salinarum

The ferredoxin of the extreme haloarchaeon Halobacterium salinarum requires high (>2 M) concentration of salt for its stability. We have used a variet y of spectroscopic probes for identifying the structural elements which nec essitate the presence of high salt for its stability. Titration of either t he fluorescence intensity of the tryptophan residues or the circular dichro ism (CD) at 217 nm with salt has identified a structural form at low (<0.1 M) concentration of salt. This structural form (L) exhibits increased solve nt exposure of W side chain(s) and decreased level of secondary structure c ompared to the native (N) protein at high concentrations of salt. The L-for m, however, contains significantly higher levels of both secondary and tert iary structures compared to the form (U) found in highly denaturing conditi ons such as 8 M urea. The structural integrity of the L-form was highly pH dependent while that of N- or U-form was not. The pH dependence of either f luorescence intensity or CD of the L-form showed the presence of two appare nt pK values: <similar to>5 and similar to 10. The structural integrity of the L-form at low (<5) pH was very similar to that of the N-form. However, titration with denaturants showed that the low pH L-form is significantly l ess stable than the N-form. The increased destabilization of the L-form wit h the increase in pH was interpreted to be due to mutual Coulombic repulsio n of carboxylate side chains (pK <approximate to> 6) and due to the disrupt ion of salt bridge(s) between ionized carboxylates and protonated amino gro ups (pK approximate to 10). Estimation of solvent accessibility of W residu es by fluorescence quenching, and measurement of decay kinetics of fluoresc ence intensity and anisotropy strongly support the above model. Polylysine interacted stoichiometrically with the L-form of ferredoxin resulting in na tivelike structure. In conclusion, our studies show that high concentration of salt stabilizes the haloarchaeal ferredoxin in two ways: (i) neutraliza tion of Coulombic repulsion among carboxyl groups of the acidic residues, a nd (ii) salting out of hydrophobic residues leading to their burial and str onger interaction.