Modeling-based control of the crystallizability of proteins in biotechnological processes

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Up to now, proper conditions for crystallization of proteins on a technical scale can only be identified empirically. Aim of the present research project is to substitute the conventional elaborate empirical approach by a modeling-based approach. During the first funding period, neutron and X-ray diffractometry and MD simulations were applied to specifically generated mutants of the exemplary protein Lactobacillus brevis alcohol dehydrogenase (LbADH). It was shown that rational protein engineering led to an improvement of protein crystallization which was mechanistically explainable. In the course of the follow-up project, a transfer of the gained knowledge to a related enzyme, Lactobacillus kefir alcohol dehydrogenase (LkADH), will be performed first. Then, it will be investigated via crystallographic and MD simulations whether mutations that had improved the crystallization of LbADH affect the inter-molecular forces during crystallization of LkADH in a similar manner. The experimental results of the crystallization behavior of LkADH and selected mutants will be used for the validation and extension of the theoretical methods. Based on this work, the gained theoretical insights into protein crystallization and its controllability are to be utilized for the first time for the de novo crystallization of two proteins that have not yet been crystallized, a dihydroxy-acid dehydratase and an ene-reductase. Homology models will be used to determine amino acid positions that can form potential crystal contacts. By applying rational protein engineering, an intensification of the interactions at the crystal contacts is subsequently to be induced. The developed MD simulation methods will be applied to predict the crystallization behavior. After experimental validation, the molecular insights into protein crystallization will be extended. The experimental evaluation will be performed by characterizing the phase behavior of proteins using high-throughput screening methods (HTS) as well as by analyzing their rheological characteristics in solution using high-frequency rheometry in order to gain a comprehensive understanding of the relationship between the viscoelastic behavior of dissolved proteins and their crystallization propensity. Finally, a process-analyzing technology (PAT) method will be developed in order to enable a real-time monitoring of the protein crystallization process via selective quantification of changes in the concentration of the target protein using UV/Vis spectroscopy. The joint research project of TUM and KIT addresses the molecular scale (protein engineering and MD simulations) as well as the microscopic scale (phase behavior and rheology). Furthermore, an analysis on the macroscopic scale (PAT) is included in order to enable an integral fundamental elucidation of basic engineering principles of technical protein crystallization.