Stabilization of the ribosomal protein S6 by trehalose is counterbalanced by the formation of a putative off-pathway species.
The effect of trehalose on folding and stability of the small ribosomal protein S6 was studied. Non-disruptive point mutations distributed along the protein structure were analyzed to characterize the stabilizing effect of trehalose and map the folding pathway of S6. On average, the stability of the wild-type and S6 mutants increases by 3 kcal/mol M trehalose. Despite the non-specific thermodynamic stabilization mechanism, trehalose particularly stabilizes the less destabilized mutants. Folding/unfolding kinetics shows clearly that trehalose induces the collapse of the unfolded state to an off-pathway intermediate with non-native diffuse contacts. This state is similar to the collapsed state induced by high concentrations of stabilizing salts, as previously reported. Although it leads to the accumulation of this off-pathway intermediate, trehalose does not change the compactness of the transition state ensemble. Furthermore, the productive folding pathway of S6 is not affected by trehalose as shown by a Phi-value analysis. The unfolded state ensemble of S6 should be more compact in the presence of trehalose and therefore destabilized due to decreased conformational entropy. Increased compaction of the unfolded state ensemble might also occur for more stable mutants of S6, thus explaining the synergistic effect of trehalose and point mutations on protein stabilization.
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Luyang
L
Chen
Gonçalo J M
GJ
Cabrita
Daniel E
DE
Otzen
Eduardo Pinho
EP
Melo
D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D038601:Ribosomal Protein S6 / Q000737:chemistry*; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000378:metabolism*; D014199:Trehalose / Q000737:chemistry*; D014466:Ultraviolet Rays
D038601:Ribosomal Protein S6; D014199:Trehalose