Linked Life Data

17348022

Source:http://linkedlifedata.com/resource/pubmed/id/17348022

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2007-4-12
pubmed:abstractText
A cell-free protein synthesis system is a powerful tool with which unnatural amino acids can be introduced into polypeptide chains. Here, the authors describe unnatural amino acid probing in a wheat germ cell-free translation system as a method for detecting the structural changes that occur in a cofactor binding protein on a conversion of the protein from an apo-form to a holo-form. The authors selected the FMN-binding protein from Desulfovibrio vulgaris as a model protein. The apo-form of the protein was synthesized efficiently in the absence of FMN. The purified apo-form could be correctly converted to the holo-form. Thus, the system could synthesize the active apo-form. Gel filtration chromatography, analytical ultracentrifugation, and circular dichroism-spectra studies suggested that the FMN-binding site of the apo-form is open as compared with the holo-form. To confirm this idea, the unnatural amino acid probing was performed by incorporating 3-azido-L-tyrosine at the Tyr35 residue in the FMN-binding site. The authors optimized three steps in their system. The introduced 3-azido-L-tyrosine residue was subjected to specific chemical modification by a fluorescein-triarylphosphine derivative. The initial velocity of the apo-form reaction was 20 fold faster than that of the holo-form, demonstrating that the Tyr35 residue in the apo-form is open to solvent.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
1097-0134
pubmed:author
pubmed:copyrightInfo
2007 Wiley-Liss, Inc.
pubmed:issnType
Electronic
pubmed:day
15
pubmed:volume
67
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
643-52
pubmed:meshHeading
pubmed:year
2007
pubmed:articleTitle
Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing.
pubmed:affiliation
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't