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Proposing Institution

Lehrstuhl für Bioverfahrenstechnik , TUM
Project Manager

Prof. Dr. Martin Zacharias
Boltzmannstr. 15
85748 Garching
The costs for purification of biopharmaceuticals via conventional chromatographic methods are high. Therefore, protein crystallization is currently being investigated as a purification method. If performed on a technical scale, this method may represent a promising low-cost alternative. However, the molecular processes for crystallization are not understood in detail. Especially, the role of hydrophobic and electrostatic interactions and their interplay with the network of hydrogen bonds on the protein interfaces are largely unknown. Up to now, proper conditions for crystallization of proteins can only be identified empirically. Therefore, aim of the present interdisciplinary project is to substitute the traditional elaborate empirical approach for identification of proper crystallization conditions by a modelling-based approach. In order to reach this aim, it is planned to perform umbrella sampling and forward flux sampling molecular dynamics (MD) simulations. Forward flux sampling is expected to reveal detailed results on the protein association process. In particular, it will predict crystal contacts and the association pathway. In addition, forward flux sampling is expected to reveal information about the crystallization kinetics. Both, forward flux and umbrella sampling will be used to evaluate the free energy profiles for the dissociation of specific protein contacts. Umbrella sampling is performed along a predefined reaction coordinate. The simulations shall help predicting conditions or suggesting directed mutagenesis for a better crystallization. Experimental protein crystallization and directed mutagenesis will be conducted at the Institute of Biochemical Engineering at TUM. The analysis of the crystals will be performed via neutron diffraction using the instrument “BioDiff” at FRM II. Recombinant lipase B from Candida antarctica and a recombinant lipase from Thermomyces lanuginosus, which are both already under experimental investigation, will be the model proteins for the simulation work. The MD simulations together with the experimental work will have a major impact on the field of protein engineering in general and protein crystallization in particular.

Impressum, Conny Wendler