Eis P S, Lakowicz J R
Department of Biological Chemistry, University of Maryland, School of Medicine, Baltimore 21201.
Biochemistry. 1993 Aug 10;32(31):7981-93. doi: 10.1021/bi00082a020.
Time-resolved frequency-domain fluorescence energy transfer measurements have been used to investigate the solution structure of a single-domain CCHH-type zinc finger peptide. These measurements reveal not only the range of accessible distances for a given donor-acceptor pair within the molecule but also the degree of conformational flexibility that occurs in solution. Two donor-acceptor (D-A)-pair zinc finger peptides have been synthesized. A single tryptophan residue located at the midpoint of the sequence was the energy donor for two different acceptors. One acceptor, attached at the amino terminus was a 5-(dimethylamino)-1-naphthalenesulfonyl (DNS) group; the second acceptor was a 7-amino-4-methyl-coumarin-3-acetyl (AMCA) group, attached to the epsilon-amino function of a carboxy-terminal lysine residue. Distance distributions and the mutual site-to-site diffusion coefficients were determined for these two D-A-labeled peptides under zinc-bound, metal-free, and denatured conditions. The D-A distance distributions determined for these two peptides under metal-free and zinc-bound conditions indicated a shorter distance and a unique conformation (narrow distribution) when metal was bound and a longer distance with greater conformational flexibility when metal ion was absent. No site-to-site diffusion was detected for the zinc-bound peptide, whereas an appreciable amount of diffusion was measured for both metal-free and denatured peptide. Anisotropy measurements on the peptides indicated increased flexibility of all regions of the peptide chain in the absence of zinc and a more compact, less flexible structure when zinc was bound. It was concluded from these results that the metal-bound conformation represents a unique, well-defined structure. Comparison of distance distributions measured for metal-free and denatured peptide indicated that there is some residual structure present in the metal-free peptide.
时间分辨频域荧光能量转移测量已被用于研究单结构域CCHH型锌指肽的溶液结构。这些测量不仅揭示了分子内给定供体-受体对可及距离的范围，还揭示了溶液中发生的构象灵活性程度。已合成了两种供体-受体（D-A）对锌指肽。位于序列中点的单个色氨酸残基是两种不同受体的能量供体。一种连接在氨基末端的受体是5-（二甲基氨基）-1-萘磺酰基（DNS）基团；第二种受体是7-氨基-4-甲基香豆素-3-乙酰基（AMCA）基团，连接到羧基末端赖氨酸残基的ε-氨基官能团上。在锌结合、无金属和变性条件下，测定了这两种D-A标记肽的距离分布和相互的位点间扩散系数。在无金属和锌结合条件下测定的这两种肽的D-A距离分布表明，结合金属时距离较短且构象独特（分布狭窄），而不存在金属离子时距离较长且构象灵活性更大。对于锌结合肽未检测到位点间扩散，而对于无金属和变性肽均测量到了可观的扩散量。对这些肽的各向异性测量表明，在没有锌的情况下肽链所有区域的灵活性增加，而结合锌时结构更紧凑、灵活性更小。从这些结果得出结论，金属结合构象代表一种独特的、明确的结构。对无金属和变性肽测量的距离分布的比较表明，无金属肽中存在一些残余结构。
