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

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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.