Double point mutant F34W/W140F of staphylococcal nuclease is in a molten globule state but highly competent to fold into a functional conformation.
The double point mutant F34W/W140F of staphylococcal nuclease was created and then characterized by far and near-UV CD, size-exclusion chromatography, ANS-binding fluorescence. The results show that this mutant has properties consistent with the classical definition of a molten globule, i.e., substantial secondary structure but no unique tight packing of tertiary structure, a relatively compact size and a larger exposed hydrophobic surface area as compared with the wild type enzyme, indicating that a molten globule can occur under physiological conditions. However, the activity assay showed that the mutant still maintains wild-type levels of activity. To further clarify the mechanism of the substrate-induced reactivation, enzymatic parameters such as K(M)(DNA), K(S)(DNA), K(M)(Ca), K(A)(Ca), K(d)(pdTp), and V(max) were determined, showing that all the parameters of this mutant are similar to those of the wild type enzyme. The results indicate that the F34W/W140F mutant has a similar substrate affinity to the wild type enzyme, and the functional conformation can be restored by substrate binding, which corresponds to the conformational adjustment capability of the mutant upon binding to ligands pdTp and Ca(2+). The severely disrupted tertiary structure and high activity of the mutant indicate that it is highly competent to fold to its functional conformation. The results suggest that the primary structure can only guide the mutant to a molten globule state and that ligand-binding causes the mutant to fold further into its functionally active conformation, indicating that ligand-binding plays an important role in protein folding and catalysis.
Li
Y
Y
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, People's Republic of China.
Jing
G
G
D000817:Anilino Naphthalenesulfonates; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000235:genetics*; D017354:Point Mutation; D011487:Protein Conformation; D017510:Protein Folding; D013329:Structure-Activity Relationship
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D008836:Micrococcal Nuclease