Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale Andrea Doria 6, 95125 Catania, Italy.
J Phys Chem B. 2010 Nov 4;114(43):13830-8. doi: 10.1021/jp1033036.
The flexible N-terminal domain of the prion protein (PrP(c)) is believed to play a pivotal role in both trafficking of the protein through the cell membrane and its pathogenic conversion into the β sheet-rich scrapie isoform (PrP(sc)). Unlike mammalian PrP(c), avian prion proteins are not known to undergo any pathogenic conformational conversions. Consequently, some critical advances in our understanding of the molecular mechanisms underlying prion pathogenesis are expected from comparative studies of the biophysical properties of the N-terminal domains of the two proteins. The present study addresses the role played by different environmental factors, i.e., copper(II), pH, and membrane-mimicking environments, in assisting the conformational preferences of huPrP60-91 and chPrP53-76, two soluble peptides encompassing the N-terminal copper(II) binding domains of the human and chicken prion proteins, respectively. Moreover, the membrane interactions of huPrP60-91, chPrP53-76, and their copper(II) complexes were evaluated by Trp fluorescence in conjunction with measurements of the variation in thermotropic properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) unilamellar vesicles. Circular dichroism experiments revealed that huPrP60-91 adopts a predominant polyproline II conformation in aqueous solution that is destabilized at basic pH or in the presence of trifluoroethanol (TFE). Unlike anionic sodium dodecyl sulfate (SDS), which seems to stabilize the polyproline II conformation further, zwitterionic dodecylphosphocholine (DPC) micelles do not affect the peptide structure. On the contrary, copper(II) promptly promotes an increase in β-turn-rich structures. Differential scanning calorimetry (DSC) and Trp fluorescence assays carried out on DPPC model membranes after incubation with huPrP60-91 showed a marked tendency of the peptide to slowly penetrate the lipid bilayer with a concomitant conformational transition toward an extended β-sheet-like structure. Such an event, which was ascribed to the hydrophobic Trp side chain residues, was shown to also depend on the level of copper(II) occupancy along the peptide. Conversely, the CD spectra of chPrP53-76 aqueous solutions indicated the presence of a mixture of random-coil/β-turn-like structures whose resulting equilibrium was influenced by SDS and copper(II) addition. Furthermore, chPrP53-76 did not exhibit any tendency to interact with model membranes in either the presence or absence of copper(II). The results reported here provide evidence of the different roles played by environmental factors in affecting the conformation and membrane activity of human and avian prion N-terminal domains.
该研究探讨了不同环境因素（即铜(II)、pH 值和模拟膜环境）在协助人源性 PrP60-91 和鸡源性 PrP53-76 两种可溶性肽的构象偏好方面所起的作用。这两种肽分别包含人源性和鸡源性朊病毒蛋白的 N 端铜(II)结合域。此外，还通过色氨酸荧光与 1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱（DPPC）单层囊泡热致性质变化的测量相结合，评估了 huPrP60-91、chPrP53-76 及其铜(II)配合物与膜的相互作用。圆二色性实验表明，huPrP60-91 在水溶液中优先采用多脯氨酸 II 构象，该构象在碱性 pH 值或三氟乙醇（TFE）存在的情况下不稳定。与似乎进一步稳定多脯氨酸 II 构象的阴离子十二烷基硫酸钠（SDS）不同，两性离子十二烷基磷酸胆碱（DPC）胶束不会影响肽结构。相反，铜(II)立即促进富含β-转角结构的增加。用 huPrP60-91 孵育 DPPC 模型膜后进行差示扫描量热法（DSC）和色氨酸荧光测定，表明肽有明显的缓慢穿透脂质双层的趋势，同时构象向伸展的β-片状结构转变。这种事件归因于疏水性色氨酸侧链残基，还取决于肽上铜(II)占据的水平。相反，chPrP53-76 水溶液的 CD 谱表明存在随机卷曲/β-转角样结构的混合物，其平衡受 SDS 和铜(II)添加的影响。此外，chPrP53-76 无论是否存在铜(II)都没有与模型膜相互作用的趋势。本研究结果提供了证据，证明环境因素在影响人源性和禽类朊病毒 N 端结构域的构象和膜活性方面发挥了不同的作用。
