Effects of heat shock protein CLPC’S ɑ4-β2 loop deletion from an alkaliphilic Bacillus lehensis G1 on its stability and activity

Abstract

Protein loops are frequently considered as critical determinants that influence not only the function but also the structure of a protein. Bacillus lehensis G1 ClpC (WT) has a four-residue insertion at the ɑ4-β2 loop, which is absent in Bacillus subtillis ClpC. To foster a deep understanding of the significance of additional residues in the structure and function of ClpC, a deletion mutation involving residues 76-79 (∆76-79) was constructed. Circular dichroism spectroscopy was used to evaluate the structural perturbations associated with the deletion. The results demonstrated that, the precise configuration of the ɑ4-β2 loop is important for maintaining the structure and function of WT. ∆76-79 leads to severe global destabilisation and unfolding of the secondary structure of the protein, which decreases ATPase activity. The optimum temperature for ∆76-79 is 25 °C, down from 45 °C for WT. The results suggest that the additional four residues at the ɑ4-β2 loop are critical for WT’s structure and function.