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Structural determinants of protein stabilization by solutes

Despite their high sequence homology, rubredoxins from Desulfovibrio gigas and D. desulfuricans are stabilized to very different extents by compatible solutes such as diglycerol phosphate, the major osmolyte in the hyperthermophilic archaeon Archaeoglobus fulgidus[Lamosa P, Burke A, Peist R, Huber R, Liu M Y, Silva G, Rodrigues‐Pousada C, LeGall J, Maycock C and Santos H (2000) Appl Environ Microbiol66, 1974–1979]. The principal structural difference between these two proteins is the absence of the hairpin loop in the rubredoxin from D. desulfuricans. Therefore, mutants of D. gigas rubredoxin bearing deletions in the loop region were constructed to investigate the importance of this structural feature on protein intrinsic stability, as well as on its capacity to undergo stabilization by compatible solutes. The three‐dimensional structure of the mutant bearing the largest deletion, Δ17|29, was determined by 1H‐NMR, demonstrating that, despite the drastic deletion, the main structural features were preserved. The dependence of the NH chemical shifts on temperature and solute concentration (diglycerol phosphate or mannosylglycerate) provide evidence of subtle conformational changes induced by the solute. The kinetic stability (as assessed from the absorption decay at 494 nm) of six mutant rubredoxins was determined at 90 °C and the stabilizing effect exerted by both solutes was assessed. The extent of protection conferred by each solute was highly dependent on the specific mutant examined: while the half‐life for iron release in the wild‐type D. gigas rubredoxin increased threefold in the presence of 0.1 m diglycerol phosphate, mutant Δ23|29 was destabilized. This study provides evidence for solute‐induced compaction of the protein structure and occurrence of weak, specific interactions with the protein surface. The relevance of these findings to our understanding of the molecular basis for protein stabilization is discussed.