四螺旋束拓扑结构对血红素结合及氧化还原性质的影响。

Effect of four helix bundle topology on heme binding and redox properties.

作者信息

Gibney B R, Rabanal F, Reddy K S, Dutton P L

机构信息

Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

出版信息

Biochemistry. 1998 Mar 31;37(13):4635-43. doi: 10.1021/bi971856s.

DOI:10.1021/bi971856s

PMID:9521784

Abstract

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 microM 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 (Em8) 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.

摘要

我们设计了两种用于构建模型的替代四螺旋束蛋白支架拓扑结构，以研究螺旋方向对我们的原型血红素蛋白模型（α-SS-α）2（先前称为H10H24 [罗伯逊，D.E.，法里德，R.S.，莫泽，C.C.，马尔霍兰，S.E.，皮迪基蒂，R.，利尔，J.D.，万德，A.J.，德格拉多，W.F.和达顿，P.L.（1994年）《自然》368，425]）的血红素结合和氧化还原性质的影响。将（α-SS-α）2的二硫键桥连的二α-螺旋单体转化为单条多肽链会导致62个氨基酸组成的螺旋-环-螺旋单体（α-l-α）内螺旋偶极和侧链的拓扑重排，该单体自组装形成（α-l-α）2。在（α-l-α）的N端添加一个半胱氨酸残基并随后氧化，得到一个126个氨基酸的单分子四螺旋束（α-l-α-SS-α-l-α）。凝胶渗透色谱表明，（α-SS-α）²和（α'-SS-α'）²（原型的一种独特结构变体），以及（α-l-α）²和（α'-l-α'）²按设计组装成不同的四螺旋束，而（α-l-α-SS-α-l-α）以单体四α-螺旋束形式洗脱。圆二色（CD）光谱证明这些肽具有高度的α-螺旋结构，并且四个血红素的掺入对二级结构的螺旋含量影响很小。通过紫外可见光谱评估了四个血红素解离常数，每种肽的解离常数范围在15 nM至25 μM之间。可见CD中出现的科顿效应表明血红素位于蛋白质结构内。通过氧化还原电位测定法确定，每种肽中四个结合血红素的平衡氧化还原中点电位（Em8）在-100至-280 mV之间。与（α-SS-α）2和双组氨酸连接的b型细胞色素一样，与（α-l-α）2和（α-l-α-SS-α-l-α）结合的血红素的血红素亲和力和光谱电化学性质相似，无论这些拓扑重排带来的整体结构变化如何。这些肽的疏水核心支持局部静电场，从而产生类似天然的血红素发色团性质（光谱学、升高的还原电位、血红素-血红素电荷相互作用以及与外源性双原子的反应性），说明了这些非天然肽在金属蛋白研究中的实用性。