Probing the Unfolding Region in a Thermolysin-like Protease by Site-Specific Immobilization
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- 作者：Johanna Mansfeld ; Gert Vriend ; Bertus Van den Burg ; Vincent G. H. Eijsink ; and Renate Ulbrich-Hofmann
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：June 29, 1999
- 年：1999
- 卷：38
- 期：26
- 页码：8240 - 8245
- 全文大小：76K
- 年卷期：v.38,no.26(June 29, 1999)
- ISSN：1520-4995
文摘
Protein stabilization by immobilization has been proposed to be most effective if the proteinis attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we havestudied 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 previousstudies had revealed which parts of the molecule are likely to be involved in the early steps of thermalunfolding. Cysteine residues were introduced by site-directed mutagenesis into various positions of acysteine-free variant of TLP-ste. The mutant enzymes were immobilized in a site-specific manner ontoActivated Thiol-Sepharose. Two mutants (T56C, S65C) having their cysteine in the proposed unfoldingregion of TLP-ste showed a 9- and 12-fold increase in half-lives at 75 
C due to immobilization. Thestabilization by immobilization was even larger (33-fold) for the T56C/S65C double mutant enzyme. Incontrast, 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 stabilizationobtained 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 regionmay be of general use for the design of strategies to stabilize proteins.
C due to immobilization. Thestabilization by immobilization was even larger (33-fold) for the T56C/S65C double mutant enzyme. Incontrast, 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 stabilizationobtained 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 regionmay be of general use for the design of strategies to stabilize proteins.