Leffers K-W, Schell J, Jansen K, Lucassen R, Kaimann T, Nagel-Steger L, Tatzelt J, Riesner D
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany.
J Mol Biol. 2004 Nov 26;344(3):839-53. doi: 10.1016/j.jmb.2004.09.071.
A series of structural intermediates in the putative pathway from the cellular prion protein PrP(C) to the pathogenic form PrP(Sc) was established by systematic variation of low concentrations (<0.1%) of the detergent sodium dodecyl sulfate (SDS) or by the interaction with the bacterial chaperonin GroEL. Most extended studies were carried out with recombinant PrP (90-231) corresponding to the amino acid sequence of hamster prions PrP 27-30. Similar results were obtained with full-length recombinant PrP, hamster PrP 27-30 and PrP(C) isolated from transgenic, non-infected CHO cells. Varying the incubation conditions, i.e. the concentration of SDS, the GroEL and GroEL/ES, but always at neutral pH and room temperature, different conformations could be established. The conformations were characterized with respect to secondary structure as determined by CD spectroscopy and to molecular mass, as determined by fluorescence correlation spectroscopy and analytical ultracentrifugation: alpha-helical monomers, soluble alpha-helical dimers, soluble but beta-structured oligomers of a minimal size of 12-14 PrP molecules, and insoluble multimers were observed. A high activation barrier was found between the alpha-helical dimers and beta-structured oligomers. The numbers of SDS-molecules bound to PrP in different conformations were determined: Partially denatured, alpha-helical monomers bind 31 SDS molecules per PrP molecule, alpha-helical dimers 21, beta-structured oligomers 19-20, and beta-structured multimers show very strong binding of five SDS molecules per PrP molecule. Binding of only five molecules of SDS per molecule of PrP leads to fast formation of beta-structures followed by irreversible aggregation. It is discussed that strongest binding of SDS has an effect identical with or similar to the interaction with GroEL thereby inducing identical or very similar transitions. The interaction with GroEL/ES stabilizes the soluble, alpha-helical conformation. The structure and their stabilities and particularly the induction of transitions by interaction of hydrophobic sites of PrP are discussed in respect to their biological relevance.
通过对低浓度（<0.1%）去污剂十二烷基硫酸钠（SDS）进行系统变化或与细菌伴侣蛋白GroEL相互作用，建立了从细胞朊蛋白PrP(C)到致病形式PrP(Sc)的假定途径中的一系列结构中间体。大多数深入研究是用与仓鼠朊病毒PrP 27 - 30氨基酸序列相对应的重组PrP（90 - 231）进行的。从转基因未感染的CHO细胞中分离的全长重组PrP、仓鼠PrP 27 - 30和PrP(C)也获得了类似结果。改变孵育条件，即SDS、GroEL和GroEL/ES的浓度，但始终保持中性pH和室温，可以建立不同的构象。通过圆二色光谱法测定二级结构以及通过荧光相关光谱法和分析超速离心法测定分子量来表征这些构象：观察到α - 螺旋单体、可溶性α - 螺旋二聚体、最小尺寸为12 - 14个PrP分子的可溶性但β - 结构寡聚体以及不溶性多聚体。在α - 螺旋二聚体和β - 结构寡聚体之间发现了一个高活化能垒。测定了不同构象下与PrP结合的SDS分子数量：部分变性的α - 螺旋单体每个PrP分子结合31个SDS分子，α - 螺旋二聚体结合21个，β - 结构寡聚体结合19 - 20个，β - 结构多聚体每个PrP分子显示出与五个SDS分子的非常强的结合。每个PrP分子仅结合五个SDS分子会导致β - 结构快速形成，随后是不可逆聚集。讨论了SDS的最强结合具有与与GroEL相互作用相同或相似的效果，从而诱导相同或非常相似的转变。与GroEL/ES的相互作用稳定了可溶性α - 螺旋构象。就其生物学相关性讨论了这些结构及其稳定性，特别是PrP疏水位点相互作用诱导的转变。
