Sasmal Sukanya, Lincoff James, Head-Gordon Teresa
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, California.
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, California; Department of Chemistry, University of California, Berkeley, Berkeley, California; Department of Bioengineering, University of California, Berkeley, Berkeley, California; Pitzer Center for Theoretical Chemistry, University of California, Berkeley, Berkeley, California.
Biophys J. 2017 Sep 5;113(5):1002-1011. doi: 10.1016/j.bpj.2017.06.067.
Paramagnetic relaxation enhancement is an NMR technique that has yielded important insight into the structure of folded proteins, although the perturbation introduced by the large spin probe might be thought to diminish its usefulness when applied to characterizing the structural ensembles of intrinsically disordered proteins (IDPs). We compare the computationally generated structural ensembles of the IDP amyloid-β42 (Aβ42) to an alternative sequence in which a nitroxide spin label attached to cysteine has been introduced at its N-terminus. Based on this internally consistent computational comparison, we find that the spin label does not perturb the signature population of the β-hairpin formed by residues 16-21 and 29-36 that is dominant in the Aβ42 reference ensemble. However, the presence of the tag induces a strong population shift in a subset of the original Aβ42 structural sub-populations, including a sevenfold enhancement of the β-hairpin formed by residues 27-31 and 33-38. Through back-calculation of NMR observables from the computational structural ensembles, we show that the structural differences between the labeled and unlabeled peptide would be evident in local residual dipolar couplings, and possibly differences in homonuclear H-H nuclear Overhauser effects (NOEs) and heteronuclear H-N NOEs if the paramagnetic contribution to the longitudinal relaxation does not suppress the NOE intensities in the real experiment. This work shows that molecular simulation provides a complementary approach to resolving the potential structural perturbations introduced by reporter tags that can aid in the interpretation of paramagnetic relaxation enhancement, double electron-electron resonance, and fluorescence resonance energy transfer experiments applied to IDPs.
顺磁弛豫增强是一种核磁共振技术，它为折叠蛋白的结构提供了重要见解，尽管大自旋探针引入的扰动可能会被认为在用于表征内在无序蛋白（IDP）的结构集合时会降低其有用性。我们将IDP淀粉样β-蛋白42（Aβ42）的计算生成结构集合与另一个序列进行比较，在该序列中，一个连接到半胱氨酸的氮氧化物自旋标记已被引入其N端。基于这种内部一致的计算比较，我们发现自旋标记不会干扰由残基16 - 21和29 - 36形成的β-发夹的特征种群，该种群在Aβ42参考集合中占主导地位。然而，标签的存在会在原始Aβ42结构亚群的一个子集中引起强烈的种群转移，包括由残基27 - 31和33 - 38形成的β-发夹增强了七倍。通过从计算结构集合中反算核磁共振可观测量，我们表明标记和未标记肽之间的结构差异在局部残余偶极耦合中会很明显，如果顺磁对纵向弛豫的贡献在实际实验中不抑制NOE强度，那么在同核H - H核Overhauser效应（NOE）和异核H - N NOE中可能也会有差异。这项工作表明，分子模拟提供了一种补充方法，用于解决报告标签引入的潜在结构扰动，这有助于解释应用于IDP的顺磁弛豫增强、双电子 - 电子共振和荧光共振能量转移实验。
