Mixed Fluorotryptophan Substitutions at the Same Residue Expand the Versatility of 19 F Protein NMR Spectroscopy.
Mixed Fluorotryptophan Substitutions at the Same Residue Expand the Versatility of 19 F Protein NMR Spectroscopy.
复制标题
同一残基的混合氟色氨酸取代扩展了 19 F 蛋白质 NMR 光谱的多功能性。
作者：
C. Kenward;K. Shin;J. Rainey
测试了应用氟NMR表征配体与膜蛋白的结合的策略，所述膜蛋白是用在吲哚环上的不同位置处被F取代的Dahans的混合物制备的。直接比较4-、5-、6-和7-氟色氨酸的19 F NMR行为作为两个重叠爱帕琳受体（AR/APJ）区段的胶束环境和片段大小的函数;一个具有单个跨膜（TM）螺旋和两个色氨酸残基，另一个具有三个TM螺旋和两个额外的色氨酸残基。化学位移，峰型，和核自旋弛豫速率被观察到作为胶束条件和F取代位置的吲哚环的函数而变化，与一个给定的残留物的胶束或溶剂的暴露是主要的区分因素。通过两种不同的肽配体（apelin-36和apela-32）生物合成制备的含5-和7-氟代甘露聚糖同种型混合物的3-TM AR片段的滴定表明，一种配体的位点特异性19 F峰强度变化，而另一种配体则没有。相比之下，两种配体扰动1 H-15 N HSQC峰模式的程度相似。因此，对于一组给定的色氨酸残基的多个氟色氨酸类型的表征显著增强了应用19 F NMR来跟踪蛋白质构象和动力学的模糊调制而不明确要求诱变或化学修饰的潜力。
The strategy of applying fluorine NMR to characterize ligand binding to a membrane protein prepared with mixtures of tryptophans substituted with F at different positions on the indole ring was tested. The 19 F NMR behavior of 4-, 5-, 6-, and 7-fluorotryptophan were directly compared as a function of both micellar environment and fragment size for two overlapping apelin receptor (AR/APJ) segments; one with a single transmembrane (TM) helix and two tryptophan residues, the other with three TM helices and two additional tryptophan residues. Chemical shifts, peak patterns, and nuclear spin relaxation rates were observed to vary as a function of micellar conditions and F substitution position in the indole ring, with the exposure of a given residue to micelle or solvent being the primary differentiating factor. Titration of the 3-TM AR segment biosynthetically prepared as a mixture of 5- and 7-fluorotryptophan-containing isoforms by two distinct peptide ligands (apelin-36 and apela-32) demonstrated site-specific 19 F peak intensity changes for one ligand but not the other. In contrast, both ligands perturbed 1 H-15 N HSQC peak patterns to a similar degree. Characterization of multiple fluorotryptophan types for a given set of tryptophan residues, thus, significantly augments the potential to apply 19 F NMR to track otherwise obscure modulation of protein conformation and dynamics without an explicit requirement for mutagenesis or chemical modification.
影响因子：
3.7
作者：
N. Zhou;Xiaoling Zhang;Xuejun Fan;E. Argyris;Jianhua Fang;E. Acheampong;G. Dubois;R. Pomerantz
通讯作者：
N. Zhou;Xiaoling Zhang;Xuejun Fan;E. Argyris;Jianhua Fang;E. Acheampong;G. Dubois;R. Pomerantz
影响因子：
2.2
作者：
Prestegard, James H.;Agard, David A.;Moremen, Kelley W.;Lavery, Laura A.;Morris, Laura C.;Pederson, Kari
通讯作者：
Pederson, Kari
DOI：
10.1073/pnas.96.24.13744
发表时间：
1999-11-23
影响因子：
11.1
作者：
Klein-Seetharaman, J;Getmanova, EV;Khorana, HG
通讯作者：
Khorana, HG