Linked Life Data

10387069

Source:http://linkedlifedata.com/resource/pubmed/id/10387069

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
26
pubmed:dateCreated
1999-7-22
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
29
pubmed:volume
38
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
8240-5
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Probing the unfolding region in a thermolysin-like protease by site-specific immobilization.
pubmed:affiliation
Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Halle, Germany. Mansfeld@biochemtech.uni-halle.de
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't