Wong C Y, Eftink M R
Department of Chemistry, University of Mississippi, University 38677, USA.
Protein Sci. 1997 Mar;6(3):689-97. doi: 10.1002/pro.5560060318.
5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogue of tryptophan that potentially can be incorporated biosynthetically into proteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor, Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of absorption spectra, we estimate approximately 95% incorporation of 5HW into position 140 of nuclease, and we estimate approximately 98% incorporation of 7AW, CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secondary structure is not changed by the tryptophan analogues. Steady-state fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluorescence intensity and anisotropy measurements indicate that the incorporated 5HW residue, like tryptophan at position 140, has a dominant rotational correlation time that is approximately the value expected for global rotation of the protein. Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.
5-羟色氨酸（5HW）和7-氮杂色氨酸（7AW）是色氨酸的类似物，它们有可能通过生物合成掺入蛋白质中，并用作研究蛋白质-DNA和蛋白质-蛋白质复合物的光谱探针。这些探针的实用性将取决于它们能够被掺入的程度以及它们对蛋白质结构和稳定性造成最小扰动的证明。为了在一种模型蛋白质中研究这些因素，我们使用含有温度敏感型λcI阻遏物的色氨酸营养缺陷型大肠杆菌表达系统，将5HW和7AW生物合成掺入葡萄球菌核酸酶A中。两种色氨酸类似物都能高效地掺入到蛋白质中。通过对吸收光谱的分析，我们估计5HW大约95%掺入到核酸酶的第140位，并且估计7AW的掺入率约为98%。核酸酶变体的圆二色光谱与含色氨酸的蛋白质相似，表明二级结构的程度没有因色氨酸类似物而改变。稳态荧光数据显示，含5HW的核酸酶的发射最大值为338nm，含7AW的核酸酶的发射最大值为355nm。时间分辨荧光强度和各向异性测量表明，掺入的5HW残基与第140位的色氨酸一样，具有占主导地位的旋转相关时间，该时间大约是蛋白质整体旋转预期的值。盐酸胍诱导的去折叠研究表明，含5HW的蛋白质的去折叠转变是两态的，其去折叠的自由能变化与含色氨酸的蛋白质相等。相比之下，盐酸胍诱导的含7AW的核酸酶的去折叠似乎显示出非两态转变，该蛋白质的表观稳定性低于色氨酸形式的蛋白质。
