Vieyto-Nuñez Julio C, Campanile Marco, Mieres-Perez Joel, Ostermeier Lena, Agar Caitlyn, Petraccone Luigi, Del Vecchio Pompea, Oliva Rosario, Winter Roland, Sanchez-Garcia Elsa
Chair of Computational Bioengineering, Department of Biochemical and Chemical Engineering, TU Dortmund, 44227 Dortmund, Germany.
Department of Chemical Sciences, University of Naples Federico II, Via Cintia 4, 80126 Naples, Italy.
JACS Au. 2025 Jul 16;5(7):3612-3624. doi: 10.1021/jacsau.5c00629. eCollection 2025 Jul 28.
Many organisms use osmolytes to protect their cells from adverse environmental conditions. Osmolytes help to stabilize the structure of proteins and to maintain their function. We studied the binding of the competitive inhibitor proflavine to α-chymotrypsin in the presence of six biologically relevant osmolytes (TMAO, glycine, sarcosine, -dimethylglycine, betaine, and glycerol). To investigate the role of osmolytes in protein-ligand binding, we estimated the relative residence times of the ligand at the catalytic site of the enzyme, using τ-random acceleration molecular dynamics and carried out extensive molecular dynamics simulations. The computational studies were complemented with UV/Vis, circular dichroism (CD) and fluorescence spectroscopy studies as well as isothermal titration calorimetry (ITC) measurements. We found that the osmolytes can modulate the interactions between proflavine and the catalytic triad of the enzyme by modifying the solvent environment at the active site. The combination of computational and experimental studies allowed identifying two main types of behavior among the osmolytes: the experimental binding constants and computed ligand residence times are higher in the presence of glycine, sarcosine, -dimethylglycine, and glycerol. By contrast, in the presence of TMAO and betaine, the computed ligand residence times and the experimental binding constants of the α-chymotrypsin-proflavine complex are lower. The strong correlation found by us between computed ligand residence times and experimentally determined binding constants in the presence of different osmolytes is particularly relevant since the identification of parameters that directly correlate to substrate binding can provide a guide for solvent selection. Indeed, on this basis, we delivered computational predictions, concerning the binding constant of proflavine to α-chymotrypsin in 0.5 M DMSO, which we experimentally corroborated. Our comprehensive study provides a molecular rationale for the pivotal role that osmolytes play in modulating ligand binding as well as key insights that could aid the design of the liquid media for biocatalytic systems.
许多生物体利用渗透溶质来保护其细胞免受不利环境条件的影响。渗透溶质有助于稳定蛋白质的结构并维持其功能。我们研究了在六种具有生物学相关性的渗透溶质（三甲胺氧化物、甘氨酸、肌氨酸、二甲基甘氨酸、甜菜碱和甘油）存在的情况下，竞争性抑制剂原黄素与α-胰凝乳蛋白酶的结合。为了研究渗透溶质在蛋白质-配体结合中的作用，我们使用τ-随机加速分子动力学估计了配体在酶催化位点的相对停留时间，并进行了广泛的分子动力学模拟。计算研究辅以紫外/可见光谱、圆二色性（CD）和荧光光谱研究以及等温滴定量热法（ITC）测量。我们发现，渗透溶质可以通过改变活性位点的溶剂环境来调节原黄素与酶催化三联体之间的相互作用。计算和实验研究相结合，使我们能够确定渗透溶质中的两种主要行为类型：在甘氨酸、肌氨酸、二甲基甘氨酸和甘油存在的情况下，实验结合常数和计算出的配体停留时间较高。相比之下，在三甲胺氧化物和甜菜碱存在的情况下，α-胰凝乳蛋白酶-原黄素复合物的计算配体停留时间和实验结合常数较低。我们发现，在不同渗透溶质存在的情况下，计算出的配体停留时间与实验测定的结合常数之间存在很强的相关性，这一点尤为重要，因为识别与底物结合直接相关的参数可以为溶剂选择提供指导。事实上，在此基础上，我们给出了关于原黄素在0.5 M二甲基亚砜中与α-胰凝乳蛋白酶结合常数的计算预测，并通过实验进行了证实。我们的综合研究为渗透溶质在调节配体结合中所起的关键作用提供了分子理论依据，同时也提供了有助于生物催化系统液体介质设计的关键见解。
