Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil.
Department of Chemistry, Yale University, New Haven, Connecticut, United States of America.
PLoS One. 2022 Apr 22;17(4):e0267536. doi: 10.1371/journal.pone.0267536. eCollection 2022.
The bi-enzyme HisF-HisH heterodimer is part of the pathway that produces histidine and purines in bacteria and lower eukaryotes, but it is absent in mammals. This heterodimer has been largely studied probing the basis of the allosteric effects and the structural stability in proteins. It is also a potential target for antibacterial drugs. In this work, we developed a simple method to evaluate changes in the affinity between HisF and HisH in the heterodimer of the bacteria Thermotoga maritima. HisH contains a single tryptophan residue, which is exposed in the free protein, but buried in the heterodimer interface. Hence, the intrinsic fluorescence maximum of this residue changes to shorter wavelengths upon dimerization. Thus, we used the fluorescence intensity at this shorter wavelength to monitor heterodimer accumulation when HisH was combined with sub-stoichiometric HisF. Under conditions where the HisF-HisH heterodimer is in equilibrium with the free states of these enzymes, when [HisH] > [HisF], we deduced a linear function connecting [HisF-HisH] to [HisF], in which the slope depends on the heterodimer dissociation constant (Kd). Based on this equation, taking fluorescence intensities as proxies of the heterodimer and HisF concentrations, we experimentally determined the Kd at four different temperatures. These Kd values were compared to those evaluated using ITC. Both methods revealed an increase in the HisF and HisH binding affinity as the temperature increases. In spite of differences in their absolute values, the Kd determined using these methods presented an evident linear correlation. To demonstrate the effectiveness of the fluorescence method we determined the effect on the Kd caused by 12 single mutations in HisF. Coherently, this test singled out the only mutation in the binding interface. In brief, the method described here effectively probes qualitative effects on the Kd, can be carried out using common laboratory equipment and is scalable.
双酶 HisF-HisH 杂二聚体是细菌和低等真核生物中产生组氨酸和嘌呤的途径的一部分，但在哺乳动物中不存在。该杂二聚体已被广泛研究，以探究变构效应和蛋白质结构稳定性的基础。它也是潜在的抗菌药物靶点。在这项工作中，我们开发了一种简单的方法来评估嗜热栖热菌（Thermotoga maritima）的 HisF-HisH 杂二聚体中 HisF 和 HisH 之间亲和力的变化。HisH 包含一个色氨酸残基，该残基在游离蛋白中暴露，但在杂二聚体界面中被掩埋。因此，该残基的固有荧光最大值在二聚化时会向较短的波长移动。因此，我们使用该较短波长的荧光强度来监测 HisH 与亚化学计量的 HisF 结合时杂二聚体的积累。在 HisF-HisH 杂二聚体与这些酶的自由态处于平衡的条件下，当 [HisH] > [HisF] 时，我们推导出一个连接 [HisF-HisH] 和 [HisF] 的线性函数，其中斜率取决于杂二聚体解离常数（Kd）。基于该方程，我们以荧光强度作为杂二聚体和 HisF 浓度的替代物，在四个不同温度下实验测定 Kd。将这些 Kd 值与 ITC 评估的值进行比较。两种方法都表明随着温度的升高，HisF 和 HisH 的结合亲和力增加。尽管绝对值不同，但两种方法测定的 Kd 值呈明显的线性相关。为了证明荧光法的有效性，我们测定了 12 个 HisF 单点突变对 Kd 的影响。一致地，该测试突出了仅在结合界面的突变。总之，本文所述的方法有效地探测了对 Kd 的定性影响，可以使用常见的实验室设备进行，并且具有可扩展性。
