Interterminal distance and flexibility of a triantennary glycopeptide as measured by resonance energy transfer.
Three geometric isomers of a single triantennary glycopeptide, each containing two fluorophores attached to terminal positions in the molecule, were used to probe distance and flexibility of the oligosaccharide in solution. A dansyl group (energy acceptor) was attached to the C6 of Gal at either position 6', 6, or 8, and a naphthyl-2-acetyl group (energy donor) was coupled to the N terminus of the Ala-Asn peptide. (formula; see text) Resonance energy-transfer measurements revealed an average distance of approximately 22, 18, and 17 A between the donor and the acceptor attached to either the 6, 8, or 6' Gal residue, respectively. The lifetime of the donor's emission was nearly a single-exponential decay of 27 ns (96%), whereas the decay of the donor with proximally attached acceptor was fit by nonlinear least-squares analysis to a multiexponential for each glycopeptide probe. Fitting with a Lorentzian function revealed spatially distinct donor/acceptor distances presumably arising from glycopeptide branch flexibility. The results suggest that the acceptor located at Gal 8 is the most rigid relative to the donor with a single population of distances centered at 18.4 A. In contrast, the acceptor attached to either Gal 6' or 6 displayed two populations of different distances from the donor. The Gal 6 isomer contained a major population with average donor/acceptor separation distance of 21.7 A and a minor population with average separation distance of 9.7 A. Similarly, the Gal 6' isomer showed a major population with donor/acceptor separation distance of 18.3 A and a minor population with separation distance of 11.7 A. These data support the earlier conclusions that the Man alpha(1----6)Man linkage found in the core pentasaccharide of all branched N-linked oligosaccharides is flexible. In addition, the data suggest that the branch containing Gal 6 is also flexible in the triantennary glycopeptide.
Rice
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KG
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Wu
R G
RG
Brand
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Lee
Y C
YC
D000818:Animals; D037263:Asialoglycoprotein Receptor; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004735:Energy Transfer; D006020:Glycopeptides / Q000737:chemistry*; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D051381:Rats; D011971:Receptors, Immunologic / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D037263:Asialoglycoprotein Receptor; D006020:Glycopeptides; D011971:Receptors, Immunologic