Kinetics of protein crystallisation in the scalable stirred crystalliser as a function of the relative molecular contact stabilities of mutants of an alcohol dehydrogenase

可扩展搅拌结晶器中蛋白质结晶动力学作为醇脱氢酶突变体的相对分子接触稳定性的函数

• 批准号: 511354413
• 负责人: Professor Dr.-Ing. Dirk Weuster-Botz
• 金额: --
• 依托单位: Lehrstuhl für Bioverfahrenstechnik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/511354413?language=en
• 关键词: Kinetics; protein; crystallisation; scalable; stirred

One reason for the low industrial use of protein crystallisation in downstream processing is the lack of crystallisability of many proteins, as they have been trained by evolution not to crystallise in order to ensure their biological functions. Our previous research therefore dealt with the rational mutagenesis of amino acid residues at the contact sites of proteins in crystals in order to be able to specifically improve the crystallisation process. Using the example of alcohol dehydrogenase from Lactobacillus brevis (LbADH), mutants that crystallised better could be identified by the targeted exchange of individual amino acids. Using molecular dynamics (MD) simulations, we were also able to show that better or reduced crystallisability of the mutants depends only on the relative strength of the interactions at the crystal contact sites. Since complete atomic MD simulations were used for this purpose, entropic and enthalpic effects are taken into account for both the solvent water and the protein in order to be able to quantitatively determine the relative molecular contact stability of protein mutants.In addition to the achievable yield in thermodynamic equilibrium, the nucleation and crystallisation rates in the scalable stirred crystalliser are particularly relevant for technical applications. The scientific question is therefore: What influence does the relative molecular contact stability have on the crystallisation kinetics of enzyme mutants in the stirred crystalliser under comparable conditions and can predictions be made about crystallisation rates compared to the native protein? These investigations are now possible for the first time, as comparable conditions can actually be realised, since only slightly modified proteins (exchange of 1 amino acid residues) can be investigated under identical conditions during saturation crystallisation. Dynamic light scattering is to be used to measure the nucleation and crystallisation kinetics, with which the aggregation of proteins or the formation of nanocrystals can be recorded dynamically from 1 - 1000 nm. Thus, the kinetics of nucleation (aggregate formation) and nanocrystal growth starting from the LbADH tetramer in solution (6-8 nm) can be dynamically recorded over a wide size range. In addition, in-situ microscopy with corresponding automated image evaluation will be used for dynamic online recording of crystal number and size distribution (< 5 µm) in the stirred crystalliser. With the help of these measurement methods, it will be possible for the first time to quantitatively investigate how the relative molecular contact stability of protein mutants influences both nucleation and crystal growth kinetics during saturation crystallisation in the stirred crystalliser under comparable conditions.

蛋白质结晶在下游加工中的工业使用率低的一个原因是许多蛋白质缺乏可结晶性，因为它们已经通过进化而被训练为不结晶以确保其生物功能。因此，我们以前的研究涉及晶体中蛋白质接触位点的氨基酸残基的合理诱变，以便能够特异性地改善结晶过程。以来自短乳杆菌的醇脱氢酶（LbADH）为例，可以通过单个氨基酸的靶向交换来鉴定结晶更好的突变体。使用分子动力学（MD）模拟，我们也能够表明，更好的或减少的突变体的结晶性仅取决于在晶体接触点的相互作用的相对强度。由于完整的原子分子动力学模拟用于此目的，考虑到熵和熵效应的溶剂水和蛋白质，以便能够定量确定蛋白质突变体的相对分子接触稳定性。除了在热力学平衡可实现的产量，在可扩展的搅拌结晶器的成核和结晶速率是特别相关的技术应用。因此，科学问题是：在可比条件下，相对分子接触稳定性对搅拌结晶器中酶突变体的结晶动力学有什么影响，并且可以预测与天然蛋白质相比的结晶速率？这些研究现在首次成为可能，因为实际上可以实现可比的条件，因为在饱和结晶期间，在相同条件下只能研究轻微修饰的蛋白质（交换1个氨基酸残基）。动态光散射用于测量成核和结晶动力学，通过该动力学，可以从1 - 1000 nm动态记录蛋白质的聚集或纳米晶体的形成。因此，从溶液中的LbADH四聚体（6-8 nm）开始的成核（聚集体形成）和微球生长的动力学可以在宽的尺寸范围内动态记录。此外，具有相应自动图像评估的原位显微镜将用于动态在线记录搅拌结晶器中的晶体数量和粒度分布（< 5 µm）。借助这些测量方法，将有可能首次定量研究蛋白质突变体的相对分子接触稳定性如何影响在可比条件下搅拌结晶器中饱和结晶期间的成核和晶体生长动力学。