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. [GRAPHICS] Resonance energy-transfer measurements revealed an average distance of approximately 22, 18, and 17 angstrom 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 angstrom. 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 angstrom and a minor population with average separation distance of 9.7 angstrom. Similarly, the Gal 6' isomer showed a major population with donor/acceptor separation distance of 18.3 angstrom and a minor population with separation distance of 11.7 angstrom. 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.