Behrends H W, Beck-Sickinger A G, Folkers G
Department of Pharmacy, Swiss Federal Institute of Technology, Zürich, Switzerland.
Eur J Biochem. 1996 Oct 1;241(1):126-32. doi: 10.1111/j.1432-1033.1996.0126t.x.
A new approach is reported that includes multiple-peptide synthesis and CD spectroscopy of overlapping peptides to evaluate the secondary structure of the vaccinia-virus thymidine kinase (TK). We divided the sequence of the vaccinia-virus TK into 82 peptides of 15 residues that overlapped by 13 residues and covered the complete sequence of vaccinia-virus TK. All peptides were synthesized by solid-phase multiple-peptide synthesis by means of the Fmoc/tert-butyl strategy. Subsequently, the secondary structure of each peptide was studied by means of CD spectroscopy in a mixture of 30% trifluoroethanol and sodium phosphate, pH 7. Secondary-structure evaluation led to determination of a vaccinia-virus-TK secondary-structure pattern. Consecutive peptides with alpha-helical content mainly showed CD spectra with increasing and decreasing Cotton effects typical of alpha-helices. This phenomenon was used to localize the helices on the sequence. In contrast, only single CD spectra with clear beta-sheet conformation, or CD spectra of mixed secondary-structure content were observed for beta-sheets. Therefore, the exact localization of beta-sheet-containing residues was deduced by comparison with isofunctional sequence-dissimilar proteins. We identified seven alpha-helices and six beta-sheet-containing regions, which we used for a secondary-structure model of the vaccinia-virus TK protein.
报道了一种新方法，该方法包括多肽合成以及对重叠肽进行圆二色光谱分析，以评估痘苗病毒胸苷激酶（TK）的二级结构。我们将痘苗病毒TK的序列分成82个由15个残基组成且彼此重叠13个残基的肽段，这些肽段覆盖了痘苗病毒TK的完整序列。所有肽段均通过Fmoc/叔丁基策略采用固相多肽合成法合成。随后，在30%三氟乙醇和磷酸钠（pH 7）的混合物中，通过圆二色光谱法研究了每个肽段的二级结构。二级结构评估确定了痘苗病毒TK的二级结构模式。主要具有α-螺旋含量的连续肽段的圆二色光谱主要显示出典型α-螺旋的科顿效应的增减。利用这一现象在序列上定位螺旋。相比之下，对于β-折叠，仅观察到具有清晰β-折叠构象的单个圆二色光谱或混合二级结构含量的圆二色光谱。因此，通过与同功能但序列不同的蛋白质进行比较，推断出含β-折叠残基的确切定位。我们鉴定出七个α-螺旋和六个含β-折叠区域，并将其用于构建痘苗病毒TK蛋白的二级结构模型。
