Tryptophan Rotamer Distribution Revealed for the α-Helix in Tear Lipocalin by Site-Directed Tryptophan Fluorescence
Tryptophan Rotamer Distribution Revealed for the α-Helix in Tear Lipocalin by Site-Directed Tryptophan Fluorescence
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DOI：
10.1021/jp309318r
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发表时间：
2012-11-15
影响因子：
3.3
通讯作者：
Glasgow, Ben J.
中科院分区：
文献类型：
--
作者：
Gasymov, Oktay K.;Abduragimov, Adil R.;Glasgow, Ben J.
旋转异构体文库是蛋白质结构确定、建模和设计的宝贵工具。利用定点色氨酸荧光(SDTF)结合旋转异构体模型研究蛋白质在溶液中的α-螺旋构象变化：探索位于人泪液Lipocalinα-螺旋片段的单个Trp突变的结构归属，以及荧光波长(Max)值，即荧光的旋转异构体模型归属。寿命符合骨架构象。通常情况下，蛋白质中的TiP荧光显示三个寿命。然而，对于α-螺旋，分配给t和g(-)旋转体的两个寿命可以令人满意地描述Trp荧光强度：衰变。由于空间位阻的限制，g(+)旋转异构体在α-螺旋中是不可行的。将荧光法得到的色氨酸旋转异构体分布与由蛋白质X射线结晶学数据得到的旋转异构体文库进行了比较。在溶剂暴露和掩埋(三级相互作用)位置，Trp旋转异构体分布不同。一种使用旋转异构体分布与SDTF(RD-SDTF)的新策略消除了常规SDTF和其他标记技术的限制，在这些标记技术中，假设站点特定的差异，例如可获得性。RD-SDTF技术不依赖于侧链的环境差异，能够潜在地检测所有侧链都暴露在溶剂中的α-螺旋结构，该技术适用于包括膜蛋白在内的各种富含α-螺旋基序的蛋白质。
Rotamer libraries are a valuable tool for protein structure determination, modeling, and design. Site-directed tryptophan fluorescence (SDTF) was used in combination with the rotamer model, for the fluorescence intensity decays to solve alpha-helical conformations of proteins in solution: Single Trp mutations located in an alpha-helical segment of human tear lipocalin Were explored for structure assignment, Along with fluorescence lambda(max) values, the rotamer model assignment of fluorescence. lifetimes fits the backbone conformation. Typically, Tip fluorescence in proteins shows three lifetimes. However, for the alpha-helix, two lifetimes assigned to t and g(-) rotamers were satisfactory to describe Trp fluorescence intensity:decays. The g(+) rotamer is not feasible in the alpha-helix due to steric restriction. Trp rotamer distributions obtained by fluorescence were compared with the rotamer library derived from X-ray crystallography data of proteins. The Trp rotamer distributions vary for solvent exposed and buried (tertiary interaction) sites. A new strategy using the rotamer distribution with SDTF (RD-SDTF) removes the limitation of regular SDTF and other labeling techniques, in which site specific differences, e.g., accessibility, are presumed. The RD-SDTF technique does not rely on environmental differences of side chains and is able to detect alpha-helical structure where all side chains are exposed to solvent Potentially, this technique is applicable to various proteins including membrane proteins, which are rich in alpha-helix motif.