Cascade blue as a donor for resonance energy transfer studies of heme-containing proteins.
Cascade blue as a donor for resonance energy transfer studies of heme-containing proteins.
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级联蓝作为含血红素蛋白质共振能量转移研究的供体。
DOI：
10.1006/abio.2001.5523
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发表时间：
2002
影响因子：
2.9
通讯作者：
A. J. Jesaitis
中科院分区：
文献类型：
--
作者：
Ross M. Taylor;B. Lin;T. R. Foubert;J. B. Burritt;J. Sunner;A. J. Jesaitis
Cascade Blue乙酰叠氮是一种胺反应性化合物，具有非常适合荧光共振能量转移（FRET）研究的光谱特性，其中血红素辅基作为受体。为了证明级联蓝血红素光谱尺的实用性，细胞色素c被用作测试案例来校准从FRET分析获得的距离测量。修饰后，化学计量标记的细胞色素c用胰蛋白酶消化，并通过基质辅助激光解吸/电离和电喷雾电离质谱法分析衍生化片段，以确定Lys 25作为共价修饰的主要位点。对衍生蛋白的FRET分析证明Cascade Blue荧光几乎完全淬灭，表明标记的赖氨酸残基位于血红素辅基的30 A内。十二烷基硫酸钠（SDS）变性导致约28%的荧光回收率，证明该供体-受体对用于评价30-90 A的距离变化的实用性。将Cascade Blue供体分子建模到细胞色素c NMR结构的Lys 25上证实了与血红素受体的距离<或=30 A，如通过FRET分析所确定的。SDS-变性状态作为延伸链的进一步建模表明最大分离距离为45 A，也与源自FRET分析的结果一致。
Cascade Blue acetyl azide is an amine reactive compound with spectral properties ideally suited for fluorescence resonance energy transfer (FRET) studies in which heme prosthetic groups serve as acceptors. To demonstrate utility of the Cascade Blue-heme spectroscopic ruler, cytochrome c was employed as a test case to calibrate distance measurements obtained from FRET analysis. Following modification, stoichiometrically labeled cytochrome c was digested with trypsin and derivatized fragments were analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry to identify Lys25 as the predominant site of covalent modification. FRET analysis on derivatized protein demonstrated nearly complete quenching of Cascade Blue fluorescence, indicating the labeled lysine residue to reside within 30 A of the heme prosthetic group. Sodium dodecyl sulfate (SDS) denaturation resulted in an approximately 28% recovery of fluorescence, demonstrating the utility of this donor-acceptor pair for evaluating distance changes of 30-90 A. Modeling the Cascade Blue donor molecule onto Lys25 of a cytochrome c NMR structure confirmed a distance of < or =30 A from the heme acceptor, as determined by FRET analysis. Further modeling of the SDS-denatured state as an extended chain suggested a maximum separation distance of 45 A, also consistent with results derived from FRET analysis.
使用荧光光谱研究异构体选择性抑制剂与前列腺素内过氧化物合酶的结合。
DOI：
10.1021/bi971691n
发表时间：
1998
期刊：
Biochemistry.
影响因子：
--
作者：
Lanzo,CA;Beechem,JM;Talley,J;Marnett,LJ
通讯作者：
Marnett,LJ
级联蓝色衍生物：水溶性、反应性、蓝色发射染料，作为荧光标记和示踪剂进行评估。
影响因子：
2.9
作者：
Whitaker,JE;Haugland,RP;Moore,PL;Hewitt,PC;Reese,M;Haugland,RP
通讯作者：
Haugland,RP
影响因子：
7.7
作者：
Clegg, Robert M.
通讯作者：
Clegg, Robert M.