Laboratório de Biofísica Teórica, Departamento de Física, Instituto de Ciências Exatas, Naturais e Educação, Universidade Federal do Triângulo Mineiro, Uberaba, MG 38064-200, Brazil.
Departamento de Matemática, Instituto de Ciências Exatas, Naturais e Educação, Universidade Federal do Triângulo Mineiro, Uberaba, MG 38064-200, Brazil.
J Chem Phys. 2022 May 21;156(19):195101. doi: 10.1063/5.0083875.
Biotin-labeled proteins are widely used as tools to study protein-protein interactions and proximity in living cells. Proteomic methods broadly employ proximity-labeling technologies based on protein biotinylation in order to investigate the transient encounters of biomolecules in subcellular compartments. Biotinylation is a post-translation modification in which the biotin molecule is attached to lysine or tyrosine residues. So far, biotin-based technologies proved to be effective instruments as affinity and proximity tags. However, the influence of biotinylation on aspects such as folding, binding, mobility, thermodynamic stability, and kinetics needs to be investigated. Here, we selected two proteins [biotin carboxyl carrier protein (BCCP) and FKBP3] to test the influence of biotinylation on thermodynamic and kinetic properties. Apo (without biotin) and holo (biotinylated) protein structures were used separately to generate all-atom structure-based model simulations in a wide range of temperatures. Holo BCCP contains one biotinylation site, and FKBP3 was modeled with up to 23 biotinylated lysines. The two proteins had their estimated thermodynamic stability changed by altering their energy landscape. In all cases, after comparison between the apo and holo simulations, differences were observed on the free-energy profiles and folding routes. Energetic barriers were altered with the density of states clearly showing changes in the transition state. This study suggests that analysis of large-scale datasets of biotinylation-based proximity experiments might consider possible alterations in thermostability and folding mechanisms imposed by the attached biotins.
生物素标记的蛋白质被广泛用作研究活细胞中蛋白质-蛋白质相互作用和接近程度的工具。蛋白质组学方法广泛采用基于蛋白质生物素化的邻近标记技术，以研究亚细胞区室中生物分子的瞬时相互作用。生物素化是一种翻译后修饰，其中生物素分子连接到赖氨酸或酪氨酸残基上。到目前为止，基于生物素的技术已被证明是有效的亲和和邻近标签工具。然而，生物素化对折叠、结合、迁移率、热力学稳定性和动力学等方面的影响仍需要进一步研究。在这里，我们选择了两种蛋白质[生物素羧基载体蛋白（BCCP）和 FKBP3]来测试生物素化对热力学和动力学性质的影响。分别使用脱辅基（无生物素）和全辅基（生物素化）蛋白结构，在广泛的温度范围内生成基于全原子结构的模型模拟。全辅基 BCCP 含有一个生物素化位点，FKBP3 被建模为最多 23 个生物素化赖氨酸。两种蛋白质的热力学稳定性通过改变它们的能量景观而发生变化。在所有情况下，在比较apo 和 holo 模拟后，在自由能曲线和折叠途径上都观察到了差异。能垒发生了变化，态密度清楚地显示了过渡态的变化。这项研究表明，基于生物素邻近实验的大规模数据集的分析可能需要考虑到连接的生物素所带来的热稳定性和折叠机制的可能改变。
