Davies Paul, Marken Frank, Salter Simon, Brown David R
Department of Biology and Biochemistry, University of Bath, Bath, UK.
Biochemistry. 2009 Mar 31;48(12):2610-9. doi: 10.1021/bi900170n.
The prion protein is a high-affinity copper binding protein that plays a role in the neurodegenerative prion diseases when it is converted into an altered isoform. The function of the protein remains controversial, but its relationship to its metallochemistry has prompted further investigation. While many researchers continue to use short peptide models for binding studies, the clear discrepancy between data obtained with such models when compared to those of full-length recombinant proteins requires clarification with this more appropriate model. Isothermal titration calorimetry was used to assess metal affinity for PrP. Using both full-length native and recombinant prion protein, we have demonstrated that the prion protein binds copper but has little affinity for other metals. Metal binding is highly pH sensitive, being optimal at pH 7.5 for copper, nickel, and zinc and at pH 5.5 for iron. Metal binding affinity for PrP was not altered by protein glycosylation. The use of suitable thermodynamic modeling reveals complex and cooperative copper binding, with evidence of negative cooperativity within the octarepeat region. Cyclic voltammetry was utilized to assess the electrochemistry of copper-charged prion protein, and we show that mPrP has a redox potential of 0.03 +/- 0.01 V versus the saturated calomel electrode at pH 7. The analysis also indicated that PrP is able to undergo reversible redox cycling with equal oxidative and reductive charges that are largely dependent on the copper bound to the octarepeat. The fifth site provides a small contribution to this redox activity, but only when the octarepeat is present. These results show conclusively that PrP can utilize copper for electron transfer, which would be expected for a radical detoxifying enzyme, and that the octarepeat region is the functional domain.
朊病毒蛋白是一种高亲和力的铜结合蛋白，当它转化为一种改变的异构体时，会在神经退行性朊病毒疾病中发挥作用。该蛋白的功能仍存在争议，但其与金属化学的关系促使了进一步的研究。虽然许多研究人员继续使用短肽模型进行结合研究，但与全长重组蛋白相比，此类模型获得的数据存在明显差异，需要用更合适的模型加以澄清。等温滴定量热法用于评估金属对朊病毒蛋白的亲和力。使用全长天然和重组朊病毒蛋白，我们证明了朊病毒蛋白结合铜，但对其他金属几乎没有亲和力。金属结合对pH高度敏感，铜、镍和锌在pH 7.5时最佳，铁在pH 5.5时最佳。蛋白质糖基化不会改变朊病毒蛋白对金属结合的亲和力。使用合适的热力学模型揭示了复杂的协同铜结合，八肽重复区域内存在负协同性的证据。循环伏安法用于评估带铜的朊病毒蛋白的电化学性质，我们表明，在pH 7时，与饱和甘汞电极相比，mPrP的氧化还原电位为0.03±0.01 V。分析还表明，朊病毒蛋白能够进行可逆的氧化还原循环，氧化和还原电荷相等，这在很大程度上取决于与八肽重复区域结合的铜。第五个位点对这种氧化还原活性贡献较小，但仅在存在八肽重复区域时才会出现。这些结果确凿地表明，朊病毒蛋白可以利用铜进行电子转移，这对于一种自由基解毒酶来说是可以预期的，并且八肽重复区域是功能域。
