Rice K G, Wu R G, Brand L, Lee Y C
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Biochemistry. 1991 Jul 9;30(27):6646-55. doi: 10.1021/bi00241a003.
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.
一种三触角糖肽的三种几何异构体，每种异构体在分子末端位置连接有两个荧光团，用于探测溶液中寡糖的距离和柔韧性。一个丹磺酰基团（能量受体）连接到Gal的C6位，位置分别为6'、6或8，一个萘-2-乙酰基（能量供体）连接到Ala-Asn肽的N端。（分子式；见正文）共振能量转移测量结果显示，分别连接到6、8或6'位Gal残基上的供体和受体之间的平均距离约为22、18和17埃。供体发射的寿命几乎是27纳秒的单指数衰减（96%），而与近端连接受体的供体的衰减通过非线性最小二乘法分析拟合为每种糖肽探针的多指数衰减。用洛伦兹函数拟合显示，可能由于糖肽分支柔韧性而产生空间上不同的供体/受体距离。结果表明，相对于供体，位于Gal 8的受体最刚性，距离集中在18.4埃的单一群体。相比之下，连接到Gal 6'或6的受体与供体显示出两个不同距离的群体。Gal 6异构体包含一个主要群体，供体/受体平均分离距离为21.7埃，一个次要群体，平均分离距离为9.7埃。同样，Gal 6'异构体显示一个主要群体，供体/受体分离距离为18.3埃，一个次要群体，分离距离为11.7埃。这些数据支持了早期的结论，即在所有分支N-连接寡糖的核心五糖中发现的Manα(1→6)Man连接是灵活的。此外，数据表明，在三触角糖肽中，含有Gal 6的分支也是灵活的。
