Effect of four helix bundle topology on heme binding and redox properties
Effect of four helix bundle topology on heme binding and redox properties
复制标题
DOI：
10.1021/bi971856s
复制
发表时间：
1998-03-31
期刊：
影响因子：
2.9
通讯作者：
Dutton, PL
中科院分区：
文献类型：
--
作者：
Gibney, BR;Rabanal, F;Dutton, PL
我们设计了两种替代的四螺旋束蛋白支架拓扑结构用于模型构建，以检查螺旋取向对我们的原型血红素蛋白模型（α-SS-α）（2）（先前描述为H10 H24）的血红素结合和氧化还原性质的影响[Robertson，D. E、法里德河美国，莫泽角，澳-地C.的方法，Mulholland，S. E、皮迪基蒂河李尔王，J.D.，Wand，A. J.，DeGrado，W. F.、和达顿，P. L.（1994）Nature 368，425]。二硫键桥连的二-α-螺旋单体（α-SS-α）（2）转化为单一多肽链导致螺旋偶极子和侧链在62个氨基酸螺旋-环-螺旋单体（α-1-α）内的拓扑学重新取向，其自缔合形成（α-1-α）（2）。将N-末端半胱氨酸残基添加至（α-1-α），随后氧化，产生126个氨基酸的单分子四螺旋束（α-1-α-SS-α-1-α）。凝胶渗透色谱法证明，（alpha-SS-alpha）（2）和（alpha '-SS-alpha'）（2）（原型的独特结构变体）以及（alpha-l-alpha）（2）和（alpha '-l-alpha'）（2）组装成设计的不同的四个螺旋束，而（alpha-l-alpha-SS-alpha-l-alpha）作为单体四个α-螺旋束洗脱。圆二色谱（CD）光谱证明，这些肽是高度α-螺旋，并纳入四个血红素的影响不大的螺旋内容的二级结构。四血红素解离常数进行了评估，通过紫外-可见光谱和范围从15 nM至25 μ M的范围内的每一种肽。可见CD中Cotton效应的存在说明血红素存在于蛋白质结构中。平衡氧化还原中点电位（E-m？(8)通过氧化还原电位测定法测定，每种肽中四种结合的血红素的电位在-100和-280 mV之间。与（α-1-α）（2）和（α-1-α-SS-α-1-α）结合的血红素的血红素亲和力和光谱电化学性质与原型（α-SS-α）（2）和双组氨酸连接的b型细胞色素相似，而不考虑这些拓扑重排引起的整体结构变化。这些肽的疏水核心支持本地静电场，这导致在nativelike血红素发色团的性质（光谱，升高的还原电位，血红素-血红素电荷相互作用，并与外源性pathologic的反应性），说明这些非天然的肽在金属蛋白的研究中的效用。
We have designed two alternative four helix bundle protein scaffold topologies for maquette construction to examine the effect of helix orientation on the heme binding and redox properties of our prototype heme protein maquette, (alpha-SS-alpha)(2), previously described as H10H24 [Robertson, D. E., Farid, R. S., Moser, C. C., Mulholland, S. E., Pidikiti, R., Lear, J. D., Wand, A. J., DeGrado, W. F., and Dutton, P. L. (1994) Nature 368, 425]. Conversion of the disulfide-bridged di-alpha-helical monomer of (alpha-SS-alpha)(2) into a single polypeptide chain results in topological reorientation of the helix dipoles and side chains within a 62 amino acid helix-loop-helix monomer, (alpha-l-alpha) which self-associates to form (alpha-l-alpha)(2). Addition of an N-terminal cysteine residue to (alpha-l-alpha) with subsequent oxidation yields a 126 amino acid single molecule four helix bundle, (alpha-l-alpha-SS-alpha-l-alpha). Gel permeation chromatography demonstrated that (alpha-SS-alpha)(2) and (alpha'-SS-alpha')(2), a uniquely structured variant of the prototype, as well as (alpha-l-alpha)(2) and (alpha'-l-alpha')(2) assemble into distinct four helix bundles as designed, whereas (alpha-l-alpha-SS-alpha-l-alpha) elutes as a monomeric four alpha-helix bundle. Circular dichroism (CD) spectroscopy proves that these peptides are highly alpha-helical, and incorporation of four hemes has little effect on the helical content of the secondary structure. Four heme dissociation constants were evaluated by UV-visible spectroscopy and ranged from the 15 nM to 25 mu M range for each of the peptides. The presence of Cotton effects in the visible CD illustrated that the hemes reside within the protein architecture. The equilibrium redox midpoint potentials (E-m?(8) of the four bound hemes in each peptide are between -100 and -280 mV, as determined by redox potentiometry. The heme affinity and spectroelectrochemical properties of the hemes bound to (alpha-l-alpha)(2) and (alpha-l-alpha-SS-alpha-l-alpha) are similar to those of the prototype, (alpha-SS-alpha)(2), and to bis-histidine ligated b-type cytochromes, regardless of the global architectural changes imposed by these topological rearrangements. The hydrophobic cores of these peptides support local electrostatic fields which result in nativelike heme chromophore properties (spectroscopy, elevated reduction potentials, heme-heme charge interaction, and reactivity with exogenous diatomics) illustrating the utility of these non-native peptides in the study of metalloproteins.