Sau A K, Chen C A, Cowan J A, Mazumdar S, Mitra S
Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai 400 005, India.
Biophys J. 2001 Oct;81(4):2320-30. doi: 10.1016/S0006-3495(01)75878-X.
Detailed circular dichroism (CD), steady-state and time-resolved tryptophan fluorescence studies on the holo- and apo- forms of high potential iron protein (HiPIP) from Chromatium vinosum and its mutant protein have been carried out to investigate conformational properties of the protein. CD studies showed that the protein does not have any significant secondary structure elements in the holo- or apo- HiPIP, indicating that the metal cluster does not have any effect on formation of secondary structure in the protein. Steady-state fluorescence quenching studies however, suggested that removal of the iron-sulfur (Fe(4)S(4)) cluster from the protein leads to an increase in the solvent accessibility of tryptophans, indicating change in the tertiary structure of the protein. CD studies on the holo- and apo- HiPIP also showed that removal of the metal prosthetic group drastically affects the tertiary structure of the protein. Time-resolved fluorescence decay of the wild type protein was fitted to a four-exponentials model and that of the W80N mutant was fitted to a three-exponentials model. The time-resolved fluorescence decay was also analyzed by maximum entropy method (MEM). The results of the MEM analysis agreed with those obtained from discrete exponentials model analysis. Studies on the wild type and mutants helped to assign the fast picosecond lifetime component to the W80 residue, which exhibits fast fluorescence energy transfer to the Fe(4)S(4) cluster of the protein. Decay-associated fluorescence spectra of each tryptophan residues were calculated from the time-resolved fluorescence results at different emission wavelengths. The results suggested that W80 is in the hydrophobic core of the protein, but W60 and W76 are partially or completely exposed to the solvent.
对来自嗜硫小红卵菌的高电位铁蛋白（HiPIP）及其突变蛋白的全酶和脱辅基形式进行了详细的圆二色性（CD）、稳态和时间分辨色氨酸荧光研究，以探究该蛋白的构象性质。CD研究表明，全酶或脱辅基HiPIP中的蛋白均没有任何显著的二级结构元件，这表明金属簇对蛋白二级结构的形成没有任何影响。然而，稳态荧光猝灭研究表明，从蛋白中去除铁硫（Fe(4)S(4)）簇会导致色氨酸的溶剂可及性增加，这表明蛋白的三级结构发生了变化。对全酶和脱辅基HiPIP的CD研究还表明，去除金属辅基会极大地影响蛋白的三级结构。野生型蛋白的时间分辨荧光衰减拟合为四指数模型，而W80N突变体的拟合为三指数模型。时间分辨荧光衰减也通过最大熵方法（MEM）进行了分析。MEM分析的结果与离散指数模型分析的结果一致。对野生型和突变体的研究有助于将快速皮秒寿命成分归因于W80残基，该残基表现出向蛋白的Fe(4)S(4)簇的快速荧光能量转移。从不同发射波长下的时间分辨荧光结果计算出每个色氨酸残基的衰减相关荧光光谱。结果表明，W80位于蛋白的疏水核心中，但W60和W76部分或完全暴露于溶剂中。
