Härd T, Sayre M H, Geiduschek E P, Kearns D R
Department of Chemistry, University of California, San Diego, La Jolla 92093-0342.
Biochemistry. 1989 Apr 4;28(7):2813-9. doi: 10.1021/bi00433a011.
We examined the fluorescence properties of a mutant TF1 protein (transcription factor 1; a member of the type II class of DNA-binding proteins, DBPII) containing tryptophan in place of phenylalanine (TF1-W61) at position 61 in the "arms" of the protein dimer. The time-resolved fluorescence (excited at 295 nm) of Trp61 decays as a double exponential with lifetimes and amplitudes that are comparable to those found in other tryptophan-containing proteins and peptides, and the time-resolved fluorescence polarization decay indicates that the tryptophan residue possesses considerable internal flexibility, in agreement with crystal studies of the homologous HU protein. The tryptophan emission is quenched when TF1-W61 binds to DNA, and equilibrium studies based on fluorescence show that the nonspecific binding affinity of the TF1-W61 mutant to DNA is similar to that of wild-type TF1. Comparison of the time-resolved fluorescence decay and steady-state fluorescence intensity reveals at least two general classes of Trp61 in the DNA complexes. One class of tryptophans is partially quenched, and the extent of quenching in the complexes with various natural DNAs and synthetic double-stranded polynucleotides correlates with the spectral overlap between tryptophan emission and DNA absorption, indicating that through-space excitation energy transfer contributes to the observed quenching. Comparisons between experimentally determined energy transfer rates and model calculations suggest that the Trp61 is located in one of the DNA grooves at a distance of less than 7.5 A from the DNA helix axis. The second class of Trp61 is "totally" quenched, and we attribute this to tryptophan residues that are in direct contact with the DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)
我们研究了一种突变型TF1蛋白（转录因子1；II类DNA结合蛋白DBPII的成员）的荧光特性，该蛋白在其二聚体“臂”的第61位含有色氨酸取代苯丙氨酸（TF1-W61）。色氨酸61的时间分辨荧光（在295nm激发）以双指数形式衰减，其寿命和振幅与其他含色氨酸的蛋白质和肽中的相当，并且时间分辨荧光偏振衰减表明色氨酸残基具有相当大的内部灵活性，这与同源HU蛋白的晶体研究结果一致。当TF1-W61与DNA结合时，色氨酸发射被淬灭，基于荧光的平衡研究表明TF1-W61突变体与DNA的非特异性结合亲和力与野生型TF1相似。对时间分辨荧光衰减和稳态荧光强度的比较揭示了DNA复合物中至少两类色氨酸61。一类色氨酸被部分淬灭，并且在与各种天然DNA和合成双链多核苷酸形成的复合物中的淬灭程度与色氨酸发射和DNA吸收之间的光谱重叠相关，表明通过空间的激发能量转移导致了观察到的淬灭。实验测定的能量转移速率与模型计算之间的比较表明，色氨酸61位于距DNA螺旋轴小于7.5埃的一个DNA沟中。第二类色氨酸61被“完全”淬灭，我们将此归因于与DNA碱基直接接触的色氨酸残基。（摘要截短于250字）
