Lebowitz M S, Pedersen P L
Laboratory for Molecular and Cellular Bioenergetics, Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Arch Biochem Biophys. 1996 Jun 15;330(2):342-54. doi: 10.1006/abbi.1996.0261.
In the absence of an electrochemical proton gradient, the F1 moiety of the mitochondrial ATP synthase catalyzes the hydrolysis of ATP. This reaction is inhibited by a natural protein inhibitor, in a process characterized by an increase in ATPase inhibition as pH is decreased from 8.0 to 6.0. In order to gain greater insight into the molecular and chemical events underlying this regulatory process, the relationships among pH, helicity of the inhibitor protein, and its capacity to inhibit F1-ATPase activity were examined. First, peptides corresponding to four regions of the 82-amino-acid inhibitor protein were chemically synthesized and assessed for both retention of secondary structure, and capacity to inhibit F1-ATPase activity. These studies showed that a region of only 24-amino-acid residues, from Phe 22 through Len 45, accounts for the inhibitory capacity of the inhibitor protein, and that retention of native helical structure in this region is not essential for inhibition. Second, three mutants (33P34, 39P40, and 43P44) of the intact inhibitor protein were prepared in which a proline residue was inserted within the inhibitory region to disrupt native helical structure. The secondary structures and inhibitory capacities of these mutants were analyzed as a function of pH. These studies revealed that, despite the initial loss of helical structure within the inhibitory region due to proline insertion, a further loss of helical structure is required to modulate inhibitory activity. These results suggest that a loss of helical structure outside the inhibitory region correlates with an increase in inhibitory capacity. Finally, two separate mutants (H48A and H55A) were prepared in which a conserved histidine residue in the wild-type inhibitor protein was replaced with an alanine. The secondary structures and inhibitory capacities of these mutants were also investigated as a function of pH. Results indicated that, although histidine residues do not directly affect the inhibitory capacity of the protein, they are important for maintaining the inhibitor protein in an inactive form at high pH. Furthermore, these results show that loss in helical structure, although correlated with an increase in inhibitory capacity, is not essential for this function. These novel experiments are consistent with a model in which the inhibitor protein is envisioned as consisting of two regions, an inhibitory region and a regulatory region. It is suggested that reduction of pH allows for the protonation of a histidine residue blocking the interaction between the two regions, thus activating the inhibitory response. The pH reduction also correlates with a partial unfolding of the protein that may either cause or result from the loss of interaction between the two helices. This unfolding may be necessary for further optimization of inhibitor function.
在没有电化学质子梯度的情况下，线粒体ATP合酶的F1部分催化ATP的水解。这种反应受到一种天然蛋白质抑制剂的抑制，在该过程中，随着pH从8.0降至6.0，ATP酶抑制作用增强。为了更深入了解这一调节过程背后的分子和化学事件，研究了pH、抑制剂蛋白的螺旋度及其抑制F1-ATP酶活性的能力之间的关系。首先，化学合成了与82个氨基酸的抑制剂蛋白的四个区域相对应的肽段，并评估了它们的二级结构保留情况以及抑制F1-ATP酶活性的能力。这些研究表明，仅24个氨基酸残基的区域，从苯丙氨酸22到亮氨酸45，决定了抑制剂蛋白的抑制能力，并且该区域天然螺旋结构的保留对于抑制作用并非必不可少。其次，制备了完整抑制剂蛋白的三个突变体（33P34、39P40和43P44），其中在抑制区域内插入了一个脯氨酸残基以破坏天然螺旋结构。分析了这些突变体的二级结构和抑制能力随pH的变化。这些研究表明，尽管由于脯氨酸插入导致抑制区域内螺旋结构最初丧失，但还需要进一步丧失螺旋结构来调节抑制活性。这些结果表明，抑制区域外螺旋结构的丧失与抑制能力的增加相关。最后，制备了两个单独的突变体（H48A和H55A），其中野生型抑制剂蛋白中一个保守的组氨酸残基被丙氨酸取代。还研究了这些突变体的二级结构和抑制能力随pH的变化。结果表明，虽然组氨酸残基不直接影响蛋白质的抑制能力，但它们对于在高pH下将抑制剂蛋白维持在无活性形式很重要。此外，这些结果表明，螺旋结构的丧失虽然与抑制能力的增加相关，但对于该功能并非必不可少。这些新实验与一个模型一致，在该模型中，抑制剂蛋白被设想为由两个区域组成，一个抑制区域和一个调节区域。有人提出，pH降低会使阻止两个区域之间相互作用的组氨酸残基质子化，从而激活抑制反应。pH降低还与蛋白质的部分解折叠相关，这可能是两个螺旋之间相互作用丧失的原因或结果。这种解折叠可能是进一步优化抑制剂功能所必需的。
