Linked Life Data

16325425

Source:http://linkedlifedata.com/resource/pubmed/id/16325425

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2006-4-3
pubmed:abstractText
Acyl carrier proteins (ACPs) are important protein cofactors in fatty acid biosynthesis, but their acylated forms have not been well-studied. To permit detailed nuclear magnetic resonance studies of acylated spinach ACP isoform I, we have developed a new expression plasmid for recombinant production of the apo-protein and modified protocols for purifying the protein product and acylating it to form acyl-ACP. To solve plasmid stability problems associated with growth in minimal media, the ampicillin resistance gene from pSACP-2a was replaced with the tetA(C) gene from pBR322. The resulting plasmid, pSACP-2t, supported overexpression of apo-ACP in Escherichia coli BL21(DE3) cells in M9 medium containing 15NH4Cl as the sole nitrogen source. Apo-ACP was purified to homogeneity by means of polyethylene glycol precipitation and anion exchange. Two in vitro synthetic routes were used to produce acyl-ACPs. In one route, apo-ACP was converted to the holo form and the acyl form by a published protocol that employs a discrete enzymatic reaction for each step. As an alternative route to produce decanoyl-ACP, apo-ACP was directly converted to the acyl form by using holo-ACP synthase along with the non-natural substrate decanoyl-CoA. Two-dimensional 1H-15N NMR spectroscopy of decanoyl-ACP and stearoyl-ACP revealed that changes in the length of the covalently attached fatty acid do not affect the secondary structure of the protein but do influence the local conformation and dynamics.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1046-5928
pubmed:author
pubmed:issnType
Print
pubmed:volume
46
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
446-55
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2006
pubmed:articleTitle
Preparation of isotopically labeled spinach acyl-acyl carrier protein for NMR structural studies.
pubmed:affiliation
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1549, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, N.I.H., Extramural