de Sá Ribeiro Fernando, Lima Luís Maurício T R
Laboratório de Biotecnologia Farmacêutica (pbiotech), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil.
Programa de Pós-Graduação em Química Biológica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil.
ACS Omega. 2025 Aug 20;10(34):38871-38881. doi: 10.1021/acsomega.5c04454. eCollection 2025 Sep 2.
The crystallographic B-factor (Bf), also known as the Debye-Waller factor (DWF) or temperature factor, relates to the mean-square displacement of the atoms (X). X may be composed of individual contributions from lattice disorder (LT), static conformational heterogeneity (H) throughout the lattice, rigid body vibration (RB), local conformational vibration (V), and zero-point atomic fluctuation (A). The Bf has been widely employed as a surrogate measure of local protein flexibility, although such relation has not been confirmed. In addition, reproducibility of the absolute B-factor is difficult to achieve, hampering the understanding of their individual contribution. Here, we report the crystallographic investigation of the enzyme-ligand complex of trypsin with benzamidine from cryo to room temperature, through a 200 K range (9-point triplicate design), by crystal stabilization with hydrophobic grease. The extent of temperature-induced conformational changes showed no connection with their respective B-factors. The B-factor variation due to temperature was constant for all atoms of the system, of about 0.005 K. The results caution against interpreting absolute, normalized, or zero-point B-factors as direct proxies for protein dynamics, which is further supported by structural analysis of data from independent groups with trypsin-benzamidine complexes obtained under dissimilar experimental conditions. The similar thermal dependence of the B-factor for all atoms of the system suggests a major contribution of this physical variable over uniform rigid body vibration.
晶体学B因子（Bf），也称为德拜-瓦勒因子（DWF）或温度因子，与原子的均方位移（X）相关。X可能由晶格无序（LT）、整个晶格中的静态构象异质性（H）、刚体振动（RB）、局部构象振动（V）和零点原子涨落（A）的单独贡献组成。尽管这种关系尚未得到证实，但Bf已被广泛用作局部蛋白质柔韧性的替代指标。此外，绝对B因子的可重复性难以实现，这妨碍了对其各自贡献的理解。在这里，我们报告了通过用疏水油脂稳定晶体，在200K范围内（9点重复设计），从低温到室温对胰蛋白酶与苯甲脒的酶-配体复合物进行的晶体学研究。温度诱导的构象变化程度与它们各自的B因子没有关联。系统中所有原子因温度引起的B因子变化是恒定的，约为0.005K。这些结果提醒人们不要将绝对、归一化或零点B因子解释为蛋白质动力学的直接代理指标，来自不同实验条件下获得的胰蛋白酶-苯甲脒复合物的独立研究小组的数据结构分析进一步支持了这一点。系统中所有原子的B因子具有相似的热依赖性，这表明这个物理变量在均匀刚体振动方面有主要贡献。
