Temperature-dependent sensitivity enhancement of solid-state NMR spectra of α-synuclein fibrils
Temperature-dependent sensitivity enhancement of solid-state NMR spectra of α-synuclein fibrils
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DOI：
10.1007/s10858-007-9189-z
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发表时间：
2007-11-01
影响因子：
2.7
通讯作者：
Rienstra, Chad M.
中科院分区：
文献类型：
--
作者：
Kloepper, Kathryn D.;Zhou, Donghua H.;Rienstra, Chad M.
α-突触核蛋白（AS）是路易体的主要纤维成分，路易体是帕金森病的病理标志。野生型人类AS和与帕金森氏病相关的三种突变形式（A53 T、A30 P和E46 K）都通过成核依赖性途径形成原纤维;然而，这些原纤维事件的生物物理学细节尚未得到很好的理解。为了阐明AS原纤维在帕金森病中的潜在作用，需要原子水平的结构洞察力。在这里，我们表明，低温采集野生型AS fibrils的魔角旋转NMR光谱大大提高了光谱灵敏度，使检测到的自旋系统的数量大大增加。在0 +/- 3摄氏度的样品温度下，交叉极化（CP）实验产生微弱的信号。较低的温度光谱（-40 +/-3 ℃）显示出数倍的信号强度，这一效应在3D N-15-C-13-C-13实验中进一步放大，这是对该样品进行主链归属所需的。因此，3D实验能够分配原纤维刚性部分中的大多数氨基酸（大约残基64至94），以及T22、V26、A27、Y39、G41、S42、H50、V52、A53、T54、V55、V63、A107、I112和S129的暂定位点特异性分配。这些信号中的大多数在0 +/- 3 ℃下的2D或3D光谱中未观察到。因此，在低温下获得的光谱允许更完整的化学位移分配。观察AS原纤维中的大多数残基是解决3D结构的重要一步。
The protein a-synuclein ( AS) is the primary fibrillar component of Lewy bodies, the pathological hallmark of Parkinson's disease. Wild-type human AS and the three mutant forms linked to Parkinson's disease (A53T, A30P, and E46K) all form fibrils through a nucleationdependent pathway; however, the biophysical details of these fibrillation events are not yet well understood. Atomic-level structural insight is required in order to elucidate the potential role of AS fibrils in Parkinson's disease. Here we show that low temperature acquisition of magic-angle spinning NMR spectra of wild type AS fibrilsgreatly enhances spectral sensitivity, enabling the detection of a substantially larger number of spin systems. At 0 +/- 3 degrees C sample temperature, cross polarization (CP) experiments yield weak signals. Lower temperature spectra (-40 +/- 3 degrees C) demonstrated several times greater signal intensity, an effect further amplified in 3D N-15-C-13-C-13 experiments, which are required to perform backbone assignments on this sample. Thus 3D experiments enabled assignments of most amino acids in the rigid part of the fibril ( approximately residues 64 to 94), as well as tentative site-specific assignments for T22, V26, A27, Y39, G41, S42, H50, V52, A53, T54, V55, V63, A107, I112, and S129. Most of these signals were not observed in 2D or 3D spectra at 0 +/- 3 degrees C. Spectra acquired at low temperatures therefore permitted more complete chemical shift assignments. Observation of the majority of residues in AS fibrils represents an important step towards solving the 3D structure.