Probing the unfolding region in a thermolysin-like protease by site-specific immobilization

Protein stabilization by immobilization has been proposed to be most effect ive if the protein is attached to the carrier at that region where unfoldin g is initiated. To probe this hypothesis, we have studied the effects of si te-specific immobilization on the thermal stability of mutants of the therm olysin-like protease from Bacillus stearothermophilus (TLP-ste). This enzym e was chosen because previous studies had revealed which parts of the molec ule are likely to be involved in the early steps of thermal unfolding. Cyst eine residues were introduced by site-directed mutagenesis into various pos itions of a cysteine-free variant of TLP-ste. The mutant enzymes were immob ilized in a site-specific manner onto Activated Thiol-Sepharose. Two mutant s (T56C, S65C) having their cysteine in the proposed unfolding region of TL P-ste showed a 9- and 12-fold increase in half-lives at 75 degrees C due to immobilization. The stabilization by immobilization was even larger (33-fo ld) for the T56C/S65C double mutant enzyme. In contrast, mutants containing cysteines in other parts of the TLP-ste molecule (N181C, S218C, T299C) sho wed only small increases in half-lives due to immobilization (maximum 2.5-f old). Thus, the stabilization obtained by immobilization was strongly depen dent on the site of attachment. It was largest when TLP-ste was fixed to th e carrier through its postulated unfolding region. The concept of the unfol ding region may be of general use for the design of strategies to stabilize proteins.