Sharp Joshua S, Guo Jun-tao, Uchiki Tomoaki, Xu Ying, Dealwis Chris, Hettich Robert L
Graduate School of Genome Science and Technology, The University of Tennessee and Oak Ridge National Laboratory, 1060 Commerce Park, Oak Ridge, TN 37830-8026, USA.
Anal Biochem. 2005 May 15;340(2):201-12. doi: 10.1016/j.ab.2005.02.005.
Photochemically generated hydroxyl radicals were used to map solvent-exposed regions in the C14S mutant of the protein Sml1p, a regulator of the ribonuclease reductase enzyme Rnr1p in Saccharomyces cerevisiae. By using high-performance mass spectrometry to characterize the oxidized peptides created by the hydroxyl radical reactions, amino acid solvent-accessibility data for native and denatured C14S Sml1p that revealed a solvent-excluding tertiary structure in the native state were obtained. The data on solvent accessibilities of various amino acids within the protein were then utilized to evaluate the de novo computational models generated by the HMMSTR/Rosetta server. The top five models initially generated by the server all disagreed with both published nuclear magnetic resonance (NMR) data and the solvent-accessibility data obtained in this study. A structural model adjusted to fit the previously reported NMR data satisfied most of the solvent-accessibility constraints. Through minor adjustment of the rotamers of two amino acid side chains for this latter structure, a model that not only provided a lower energy conformation but also completely satisfied previously reported data from NMR and tryptophan fluorescence measurements, in addition to the solvent-accessibility data presented here, was generated.
光化学产生的羟基自由基被用于绘制酿酒酵母中核糖核酸还原酶Rnr1p的调节蛋白Sml1p的C14S突变体中溶剂暴露区域的图谱。通过使用高效质谱来表征由羟基自由基反应产生的氧化肽段，获得了天然和变性C14S Sml1p的氨基酸溶剂可及性数据，这些数据揭示了天然状态下的一种排除溶剂的三级结构。然后利用蛋白质内各种氨基酸的溶剂可及性数据来评估由HMMSTR/Rosetta服务器生成的从头计算模型。服务器最初生成的前五个模型均与已发表的核磁共振（NMR）数据以及本研究中获得的溶剂可及性数据不一致。一个根据先前报道的NMR数据进行调整的结构模型满足了大部分溶剂可及性限制。通过对该后一种结构的两个氨基酸侧链的旋转异构体进行微调，生成了一个不仅提供了较低能量构象，而且除了此处呈现的溶剂可及性数据外，还完全符合先前报道的NMR和色氨酸荧光测量数据的模型。
