Linked Life Data

17091334

Source:http://linkedlifedata.com/resource/pubmed/id/17091334

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
2006-11-22
pubmed:abstractText
A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H2O/50%D2O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a 13C'-coupled 2D 15N-1H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH2 groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0925-2738
pubmed:author
pubmed:issnType
Print
pubmed:volume
36
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
205-14
pubmed:dateRevised
2007-12-3
pubmed:meshHeading
pubmed:year
2006
pubmed:articleTitle
Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY.
pubmed:affiliation
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA. liua@msu.edu
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't, Evaluation Studies, Research Support, N.I.H., Extramural