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

下游加工中蛋白质结晶的工业使用率较低的一个原因是许多蛋白质缺乏结晶性，因为它们被进化训练成不结晶，以确保它们的生物功能。因此，我们以前的研究涉及到蛋白质在晶体中接触位置的氨基酸残基的合理突变，以便能够具体地改善结晶过程。以短乳杆菌酒精脱氢酶(LbADH)为例，通过单个氨基酸的靶向交换，可以识别结晶更好的突变体。利用分子动力学(MD)模拟，我们还能够证明突变体结晶性的好坏只取决于晶体接触位置上相互作用的相对强度。由于使用了完整的原子分子动力学模拟，为了能够定量地确定蛋白质突变体的相对分子接触稳定性，考虑了溶剂水和蛋白质的熵和焓效应。除了热力学平衡中可获得的产率外，可伸缩搅拌结晶器中的成核和结晶速率对于技术应用尤其重要。因此，科学问题是：在可比较的条件下，相对分子接触稳定性对酶突变体在搅拌结晶器中的结晶动力学有什么影响，是否可以预测与天然蛋白质相比的结晶速度？这些研究现在首次成为可能，因为实际上可以实现可比较的条件，因为在饱和结晶过程中，只有轻微修饰的蛋白质(1个氨基酸残基的交换)可以在相同的条件下进行研究。动态光散射是用来测量成核和结晶动力学的，用它可以在1-1000 nm范围内动态记录蛋白质的聚集或纳米晶的形成。因此，LbADH四聚体在溶液中(6-8 nm)的成核(聚集体形成)和纳米晶体生长的动力学可以在很大的尺寸范围内动态记录。此外，现场显微镜和相应的自动图像评估将用于动态在线记录搅拌结晶器中的晶体数量和尺寸分布(&lt；5微米)。借助于这些测量方法，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在可比条件下的搅拌结晶器中饱和结晶过程中影响成核和晶体生长动力学。

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.