Department of Physics, University of Houston, Houston, Texas.
Department of Neurobiology and Anatomy, University of Texas, Health Science Center at Houston, Houston, Texas.
Biophys J. 2020 Apr 7;118(7):1665-1678. doi: 10.1016/j.bpj.2020.02.015. Epub 2020 Feb 25.
We have developed a computational method of atomistically refining the structural ensemble of intrinsically disordered peptides (IDPs) facilitated by experimental measurements using circular dichroism spectroscopy (CD). A major challenge surrounding this approach stems from the deconvolution of experimental CD spectra into secondary structure features of the IDP ensemble. Currently available algorithms for CD deconvolution were designed to analyze the spectra of proteins with stable secondary structures. Herein, our work aims to minimize any bias from the peptide deconvolution analysis by implementing a non-negative linear least-squares fitting algorithm in conjunction with a CD reference data set that contains soluble and denatured proteins (SDP48). The non-negative linear least-squares method yields the best results for deconvolution of proteins with higher disordered content than currently available methods, according to a validation analysis of a set of protein spectra with Protein Data Bank entries. We subsequently used this analysis to deconvolute our experimental CD data to refine our computational model of the peptide secondary structure ensemble produced by all-atom molecular dynamics simulations with implicit solvent. We applied this approach to determine the ensemble structures of a set of short IDPs, that mimic the calmodulin binding domain of calcium/calmodulin-dependent protein kinase II and its 1-amino-acid and 3-amino-acid mutants. Our study offers a, to our knowledge, novel way to solve the ensemble secondary structures of IDPs in solution, which is important to advance the understanding of their roles in regulating signaling pathways through the formation of complexes with multiple partners.
我们开发了一种计算方法，可以通过使用圆二色性光谱（CD）进行的实验测量来原子级细化无规卷曲肽（IDP）的结构集合。该方法面临的主要挑战源于将实验 CD 光谱解卷积为 IDP 集合的二级结构特征。目前可用于 CD 解卷积的算法是为分析具有稳定二级结构的蛋白质的光谱而设计的。在此，我们的工作旨在通过实施非负线性最小二乘拟合算法以及包含可溶性和变性蛋白质（SDP48）的 CD 参考数据集，最大程度地减少肽解卷积分析中的任何偏差。根据一组具有蛋白质数据库条目（Protein Data Bank）的蛋白质光谱的验证分析，非负线性最小二乘方法比当前可用的方法更适合于解卷积具有更高无序含量的蛋白质。我们随后使用此分析方法对我们的实验 CD 数据进行解卷积，以细化通过隐溶剂全原子分子动力学模拟产生的肽二级结构集合的计算模型。我们应用此方法来确定一组短 IDP 的集合结构，这些 IDP 模拟钙/钙调蛋白依赖性蛋白激酶 II 的钙调蛋白结合域及其 1 个氨基酸和 3 个氨基酸突变体。我们的研究提供了一种，据我们所知，解决溶液中 IDP 集合二级结构的新方法，这对于理解它们通过与多个伴侣形成复合物来调节信号通路的作用很重要。
