Analysis and optimization of saturation transfer difference NMR experiments designed to map early self-association events in amyloidogenic peptides
Analysis and optimization of saturation transfer difference NMR experiments designed to map early self-association events in amyloidogenic peptides
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DOI：
10.1021/jp7118718
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发表时间：
2008-05-08
影响因子：
3.3
通讯作者：
Melacini, Giuseppe
中科院分区：
文献类型：
--
作者：
Huang, Hao;Milojevic, Julijana;Melacini, Giuseppe
饱和迁移差（STD）方法是近年来发展起来的一种在残基和原子分辨率上进行淀粉样蛋白自识别定位的有效方法。尽管显着的潜力，STD的方法进行低聚物/单体（O/M）平衡的系统，在STD实验的这种新的应用程序中产生的可能的工件的系统分析仍然缺乏。在这里，我们分析了STD方法适用于O/M肽，我们已经确定了三个主要来源的可能的偏见：偏移效应，单体内交叉弛豫，和部分自旋扩散内的低聚物。为了定量评估这些文物的目的，我们采用了一种比较的方法，依赖于1-D和2-D STD数据采集在不同的饱和频率的样品具有不同的肽浓度和过滤状态。该伪影评价方案应用于A β（12-28）模型系统，所有三种类型的伪影似乎都会影响测量的STD光谱。此外，我们提出了一种方法，以尽量减少这些文物中的H α STD分布用于获得肽的自我识别图在残基分辨率的偏见。这种方法依赖于平均的STD数据集在不同的饱和频率下获得，并提供与那些独立地通过其他NMR脉冲序列，探针寡聚化，如非选择性非共振弛豫实验获得的结果。人工制品的评价协议和多个频率平均策略，这里提出的是一般效用的不断增长的家族的淀粉样蛋白生成肽，因为它们提供了一个可靠的分析STD光谱的多肽自我识别表位。
Saturation transfer difference (STD) methods recently have been proposed to be a promising tool for self-recognition mapping at residue and atomic resolution in amyloidogenic peptides. Despite the significant potential of the STD approach for systems undergoing oligomer/monomer (O/M) equilibria, a systematic analysis of the possible artifacts arising in this novel application of STD experiments is still lacking. Here, we have analyzed the STD method as applied to O/M peptides, and we have identified three major sources of possible biases: offset effects, intramonomer cross-relaxation, and partial spin-diffusion within the oligomers. For the purpose of quantitatively assessing these artifacts, we employed a comparative approach that relies on 1-D and 2-D STD data acquired at different saturation frequencies on samples with different peptide concentrations and filtration states. This artifact evaluation protocol was applied to the A beta(12-28) model system, and all three types of artifacts appear to affect the measured STD spectra. In addition, we propose a method to minimize the biases introduced by these artifacts in the H alpha STD distributions used to obtain peptide self-recognition maps at residue resolution. This method relies on the averaging of STD data sets acquired at different saturation frequencies and provides results comparable to those independently obtained through other NMR pulse sequences that probe oligomerization, such as nonselective off-resonance relaxation experiments. The artifact evaluation protocol and the multiple frequencies averaging strategy proposed here are of general utility for the growing family of amyloidogenic peptides, as they provide a reliable analysis of STD spectra in terms of polypeptide self-recognition epitopes.