Calorimetric investigation of copper binding in the N-terminal region of the prion protein at low copper loading: evidence for an entropically favorable first binding event.
Calorimetric investigation of copper binding in the N-terminal region of the prion protein at low copper loading: evidence for an entropically favorable first binding event.
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DOI：
10.1021/ic502014x
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发表时间：
2015-01-20
影响因子：
4.6
通讯作者：
Burns, Colin S.
中科院分区：
文献类型：
--
作者：
Gogineni, Devi Praneetha;Spuches, Anne M.;Burns, Colin S.
虽然朊病毒蛋白的Cu 2+结合位点已经被很好地研究，当蛋白被Cu 2+完全饱和时，Cu 2+负载机制才刚刚开始进入视野。由于在低和中等Cu 2+占有率的Cu 2+结合模式必然代表最高亲和力的结合模式，这些很可能是在生理条件下填充，因此必须表征它们，以便更好地理解铜-朊病毒相互作用的生物学功能。除了结合亲和力数据，几乎没有其他热力学参数（例如，ΔH和ΔS），从而留下了未确定的决定Cu 2+与朊病毒蛋白结合的自由能的熵和熵因子。在这项研究中，等温滴定量热法（ITC）用于定量Cu 2+与肽PrP（23-28，57-98）结合的热力学参数（K，ΔG，ΔH和TΔS），该肽包含全长PrP与Cu 2+结合相关的大部分残基。使用缓冲液N-（2-乙酰氨基）-氨基乙磺酸（ACES）（其也是充分表征的Cu 2+螯合剂）允许分离两个最高亲和力结合事件。圆二色光谱被用来表征不同的结合模式作为添加的Cu 2+的函数。ITC测定的Kd值（7和380 nM）与其他人报告的值完全一致。第一个结合事件显著受益于正熵，而第二个结合事件是由熵驱动的。与阿尔茨海默病有关的Aβ肽与Cu 2+结合的热力学值与朊病毒蛋白的热力学值惊人地相似。采用等温滴定量热法研究了朊病毒蛋白与Cu ~（2+）结合的两个最高亲和力的热力学过程（K、ΔG、ΔH和TΔS）。PrP肽（23−28，57−98）被用作金属结合区的模型系统。第一个结合事件的Kd为7 nM，是熵驱动的（+ΔS），而第二个结合事件的Kd为380 nm，是熵驱动的（-ΔH）。
Although the Cu2+-binding sites of the prion protein have been well studied when the protein is fully saturated by Cu2+, the Cu2+-loading mechanism is just beginning to come into view. Because the Cu2+-binding modes at low and intermediate Cu2+ occupancy necessarily represent the highest-affinity binding modes, these are very likely populated under physiological conditions, and it is thus essential to characterize them in order to understand better the biological function of copper–prion interactions. Besides binding-affinity data, almost no other thermodynamic parameters (e.g., ΔH and ΔS) have been measured, thus leaving undetermined the enthalpic and entropic factors that govern the free energy of Cu2+ binding to the prion protein. In this study, isothermal titration calorimetry (ITC) was used to quantify the thermodynamic parameters (K, ΔG, ΔH, and TΔS) of Cu2+ binding to a peptide, PrP(23–28, 57–98), that encompasses the majority of the residues implicated in Cu2+ binding by full-length PrP. Use of the buffer N-(2-acetomido)-aminoethanesulfonic acid (ACES), which is also a well-characterized Cu2+ chelator, allowed for the isolation of the two highest affinity binding events. Circular dichroism spectroscopy was used to characterize the different binding modes as a function of added Cu2+. The Kd values determined by ITC, 7 and 380 nM, are well in line with those reported by others. The first binding event benefits significantly from a positive entropy, whereas the second binding event is enthalpically driven. The thermodynamic values associated with Cu2+ binding by the Aβ peptide, which is implicated in Alzheimer’s disease, bear striking parallels to those found here for the prion protein. The thermodynamics (K, ΔG, ΔH, and TΔS) of the two highest affinity Cu2+-binding events of the prion protein were investigated using isothermal titration calorimetry. Peptide PrP(23−28, 57−98) was used as a model system for the metal-binding region. The first binding event had a Kd of 7 nM and was entropically driven (+ΔS), whereas the second binding event had a Kd of 380 nm and was enthalpically driven (−ΔH).
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