期刊
BIOPHYSICAL JOURNAL
卷 91, 期 5, 页码 1952-1959出版社
CELL PRESS
DOI: 10.1529/biophysj.105.079913
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资金
- NCRR NIH HHS [P41 RR005351, RR05351] Funding Source: Medline
- NIGMS NIH HHS [R01 GM033225, GM33225, R37 GM033225] Funding Source: Medline
Structural characterization of biomolecules by NMR methods frequently requires the enrichment of magnetically active isotopes at particular molecular sites. Introduction is usually achieved biosynthetically through the use of bacterial cultures grown on isotopically enriched media, but for certain types of molecules-cell-surface carbohydrates of mammalian origin, for example - this is not practical. Here we explore a means of introducing C-13-enriched sites, postisolation in natural carbohydrate products, and illustrate an ability to acquire sufficient information to select appropriate conformational models from among energetically allowed sets. The application presented involves replacement of native N-acetyl groups with C-13-labeled acetyl groups in a simple disaccharide derivative, ( GlcNAc)(2)-OBu, or O-butyl-chitobiose. The assignment of the two acetyl groups introduced is based on a novel combination of NMR and mass spectrometry data. Structural information is obtained from chemical shift anisotropy offsets of C-13 carbonyl resonances and C-13-C-13 dipolar couplings between the labeled methyl and carbonyl carbons of the acetyl groups. Although the application is to a relatively simple system, it lays the groundwork for application to biologically important complex carbohydrate systems.
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