Probing the unfolding region in a thermolysin-like protease by site-specific immobilization.
Protein stabilization by immobilization has been proposed to be most effective if the protein is attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we have studied the effects of site-specific immobilization on the thermal stability of mutants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). This enzyme was chosen because previous studies had revealed which parts of the molecule are likely to be involved in the early steps of thermal unfolding. Cysteine residues were introduced by site-directed mutagenesis into various positions of a cysteine-free variant of TLP-ste. The mutant enzymes were immobilized in a site-specific manner onto Activated Thiol-Sepharose. Two mutants (T56C, S65C) having their cysteine in the proposed unfolding region of TLP-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-fold) for the T56C/S65C double mutant enzyme. In contrast, mutants containing cysteines in other parts of the TLP-ste molecule (N181C, S218C, T299C) showed only small increases in half-lives due to immobilization (maximum 2.5-fold). Thus, the stabilization obtained by immobilization was strongly dependent on the site of attachment. It was largest when TLP-ste was fixed to the carrier through its postulated unfolding region. The concept of the unfolding region may be of general use for the design of strategies to stabilize proteins.
Mansfeld
J
J
Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Halle, Germany. Mansfeld@biochemtech.uni-halle.de
Vriend
G
G
Van den Burg
B
B
Eijsink
V G
VG
Ulbrich-Hofmann
R
R
D003545:Cysteine / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000138:chemical synthesis / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature; D013820:Thermolysin / Q000737:chemistry
D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases; D013820:Thermolysin; D003545:Cysteine