用于表征模型肽 - DNA 相互作用的傅里叶变换红外光谱法。

Fourier transform infrared spectroscopy for the characterization of a model peptide-DNA interaction.

作者信息

Dev S B, Walters L

机构信息

Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge 02139.

出版信息

Biopolymers. 1990 Jan;29(1):289-99. doi: 10.1002/bip.360290131.

DOI:10.1002/bip.360290131

PMID:2328291

Abstract

To better understand the structural basis of protein-DNA interactions, the conformational changes that accompany these interactions need to be described. In order to develop a methodological approach to this problem, Fourier transform infrared spectroscopy (FTIR) with derivative resolution enhancement has been used to identify conformational changes that occur when a 29-residue synthetic peptide binds nonspecifically to heterogeneous cellular DNA in aqueous solution. The peptide sequence was chosen de novo, in order to rationally design a peptide model that would allow the relationship between DNA binding and the stability of protein secondary structure to be studied. Peptide at a concentration of 100-200 microM produces 50% saturation of heterogeneous phage DNA sequences as well as of short synthetic oligonucleotides. FTIR spectra reveal significant changes in peptide and DNA upon binding. Second-derivative spectra resolve the amide I band of native peptide into components located at 1627 (beta-strand), 1658 (alpha-helix), and 1681 (turn or beta-strand) cm-1, with a distinct shoulder at 1647 cm-1 (disordered structure). Assignment of the 1681 cm-1 vibration to a turn conformation is supported by uv CD studies, which indicate significant amounts of turn structure in unbound peptide. Ultraviolet CD also confirms the existence of disordered and beta-strand regions in the free peptide. Upon interacting with DNA the band at 1681 cm-1 (turn) is no longer seen; a new band appears at 1675 cm-1; the 1627 cm-1 band (beta-strand) is considerably reduced in intensity; the position of the alpha-helical (1658 cm-1) component remains unchanged; the shoulder at 1647 cm-1 (disorder) disappears. The new vibration at 1675 cm-1 is characteristic of beta-strand structures. The asymmetric stretch (vAS) of the DNA phosphates shifts from 1223 (unbound) to 1229 cm-1 (bound); the relative intensities of vAS and the PO2- symmetric stretch (vS) are altered upon peptide binding.(ABSTRACT TRUNCATED AT 400 WORDS)

摘要

为了更好地理解蛋白质 - DNA 相互作用的结构基础，需要描述伴随这些相互作用的构象变化。为了开发解决此问题的方法，具有导数分辨率增强功能的傅里叶变换红外光谱（FTIR）已被用于识别当一个 29 个残基的合成肽在水溶液中与异质细胞 DNA 非特异性结合时发生的构象变化。肽序列是从头选择的，以便合理设计一个肽模型，用于研究 DNA 结合与蛋白质二级结构稳定性之间的关系。浓度为 100 - 200 microM 的肽会使异质噬菌体 DNA 序列以及短合成寡核苷酸产生 50% 的饱和度。FTIR 光谱显示结合后肽和 DNA 有显著变化。二阶导数光谱将天然肽的酰胺 I 带解析为位于 1627（β - 链）、1658（α - 螺旋）和 1681（转角或β - 链）cm⁻¹ 的成分，在 1647 cm⁻¹ 处有一个明显的肩峰（无序结构）。紫外 CD 研究支持将 1681 cm⁻¹ 的振动归属于转角构象，这表明未结合肽中有大量的转角结构。紫外 CD 还证实了游离肽中存在无序和β - 链区域。与 DNA 相互作用后，1681 cm⁻¹（转角）处的谱带不再出现；在 1675 cm⁻¹ 处出现一个新谱带；1627 cm⁻¹（β - 链）谱带的强度大幅降低；α - 螺旋（1658 cm⁻¹）成分的位置保持不变；1647 cm⁻¹（无序）处的肩峰消失。1675 cm⁻¹ 处的新振动是β - 链结构的特征。DNA 磷酸基团的不对称伸缩振动（vAS）从 1223（未结合）移至 1229 cm⁻¹（结合）；肽结合后 vAS 和 PO₂⁻ 对称伸缩振动（vS）的相对强度发生改变。（摘要截取自 400 字）