Department of Biophysics, UT Southwestern Medical Center, Dallas, Texas, 75390-8816, USA.
Center for Alzheimer's and Neurodegenerative Disease, UT Southwestern Medical Center, Dallas, Texas, 75390, USA.
Protein Sci. 2023 May;32(5):e4628. doi: 10.1002/pro.4628.
Protein regions which are intrinsically disordered, exist as an ensemble of rapidly interconverting structures. Cooling proteins to cryogenic temperatures for dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR studies suspends most of the motions, resulting in peaks that are broad but not featureless. To demonstrate that detailed conformational restraints can be retrieved from the peak shapes of frozen proteins alone, we developed and used a simulation framework to assign peak features to conformers in the ensemble. We validated our simulations by comparing them to spectra of α-synuclein acquired under different experimental conditions. Our assignments of peaks to discrete dihedral angle populations suggest that structural constraints are attainable under cryogenic conditions. The ability to infer ensemble populations from peak shapes has important implications for DNP MAS NMR studies of proteins with regions of disorder in living cells because chemical shifts are the most accessible measured parameter.
无规卷曲的蛋白质区域以快速转换结构的集合形式存在。将蛋白质冷却至低温以进行动态核极化（DNP）魔角旋转（MAS）NMR 研究，可使大多数运动处于暂停状态，从而导致峰宽但非无特征。为了证明仅从冷冻蛋白质的峰形就可以获得详细的构象约束，可以开发并使用模拟框架将峰特征分配给集合中的构象。通过将模拟结果与在不同实验条件下获得的α-突触核蛋白的光谱进行比较，对我们的模拟进行了验证。我们将峰分配给离散的二面角群体的分配表明，在低温条件下可以获得结构约束。从峰形推断集合群体的能力对活细胞中无序区域的蛋白质的 DNP MAS NMR 研究具有重要意义，因为化学位移是最易测量的参数。
