Probing structural differences between PrPC and PrPSc by surface nitration and acetylation: evidence of conformational change in the C-terminus
Probing structural differences between PrPC and PrPSc by surface nitration and acetylation: evidence of conformational change in the C-terminus
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通过表面硝化和乙酰化探索 PrPC 和 PrPSc 之间的结构差异：C 末端构象变化的证据
DOI：
10.1021/bi102073j
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发表时间：
2011-06-07
期刊：
影响因子：
2.9
通讯作者：
Requena, Jesus R.
中科院分区：
文献类型：
--
作者：
Gong, Binbin;Ramos, Adriana;Requena, Jesus R.
我们使用两种化学修饰剂，四硝基甲烷(TNM)和乙酸酐(Ac2O)，分别针对酪氨酸和赖氨酸残基，修饰重组叙利亚仓鼠PrP(90-231)[rSHaPrP(90-231)]和SHaPrP 27-30，PrPSc的抗蛋白酶K核心是从感染瘙痒病的叙利亚地鼠脑中分离出来的。我们的目标是找到构象变化的位置。用胰酶或胰凝乳酶对修饰蛋白进行凝胶内消化，然后用质谱仪(MALDI-TOF)进行分析。观察到化学反应活性的几个不同之处。对于TNM，最显著的反应性差异涉及多肽E-221-R-229(包含Y-225和Y-226)，它在rSHaPrP(90-231)中比在SHaPrP 27-30中被更广泛的修饰；含有Y-128的H-111-R-136在rSHaPrP(90-231)中也被更多地修饰。相反，Y-149-R-151、Y-157-R-164和R-151-Y-162在SHaPrP 27-30中经历了更广泛的修饰。在rSHaPrP(90-231)和SHaPrP 27-30中，乙酸酐修饰了非常广泛的G(90)-K-106肽，含有K-101，K-104，K-106和氨基末端。这些结果表明：(1)SHaPrP 27-30在C-末端区域与rSHaPrP(90-231)显示了重要的构象差异，导致Y-225和Y-226在后一种构象中失去了溶剂可及性；因为其他结果表明保存了两个C-末端螺旋，这可能意味着它们紧密地堆积在SHaPrP 27-30中。(2)另一方面，在SHaPrP 27-30中，约Y-149-R-164伸展中包含的酪氨酸更容易获得，这表明在α-螺旋HI和rSHaPrP(90-231)的短β-折叠中发生了重排。(3)SHaPrP 27-30的氨基末端区域很容易接近。这些数据应有助于验证和构建PrPSc的结构模型。
We used two chemical modifiers, tetranitromethane (TNM) and acetic anhydride (Ac2O), which specifically target accessible tyrosine and lysine residues, respectively, to modify recombinant Syrian hamster PrP(90-231) [rSHaPrP(90-231)] and SHaPrP 27-30, the proteinase K-resistant core of PrPSc isolated from brain of scrapie-infected Syrian hamsters. Our aim was to find locations of conformational change. Modified proteins were subjected to in-gel proteolytic digestion with trypsin or chymotrypsin and subsequent analysis by mass spectrometry (MALDI-TOF). Several differences in chemical reactivity were observed. With TNM, the most conspicuous reactivity difference seen involves peptide E-221-R-229 (containing Y-225 and Y-226), which in rSHaPrP(90-231) was much more extensively modified than in SHaPrP 27-30; peptide H-111-R-136, containing Y-128, was also more modified in rSHaPrP(90-231). Conversely, peptides Y-149-R-151, Y-157-R-164, and R-151-Y-162 suffered more extensive modification in SHaPrP 27-30. Acetic anhydride modified very extensively peptide G(90)-K-106, containing K-101, K-104, K-106, and the amino terminus, in both rSHaPrP(90-231) and SHaPrP 27-30. These results suggest that (1) SHaPrP 27-30 exhibits important conformational differences in the C-terminal region with respect to rSHaPrP(90-231), resulting in the loss of solvent accessibility of Y-225 and Y-226, very solvent-exposed in the latter conformation; because other results suggest preservation of the two C-terminal helices, this might mean that these are tightly packed in SHaPrP 27-30. (2) On the other hand, tyrosines contained in the stretch spanning approximately Y-149-R-164 are more accessible in SHaPrP 27-30, suggesting rearrangements in alpha-helix HI and the short beta-sheet of rSHaPrP(90-231). (3) The amino-terminal region of SHaPrP 27-30 is very accessible. These data should help in the validation and construction of structural models of PrPSc.