Abstract
Protein crystallization can function as an effective method for protein purification or formulation. Such an application requires a comprehensive understanding of the intermolecular protein–protein interactions that drive and stabilize protein crystal formation to ensure a reproducible process. Using alcohol dehydrogenase from Lactobacillus brevis (LbADH) as a model system, we probed in our combined experimental and computational study the effect of residue substitutions at the protein crystal contacts on the crystallizability and the contact stability. Increased or decreased contact stability was calculated using molecular dynamics (MD) free energy simulations and showed excellent qualitative correlation with experimentally determined increased or decreased crystallizability. The MD simulations allowed us to trace back the changes to their physical origins at the atomic level. Engineered charge–charge interactions as well as engineered hydrophobic effects could be characterized and were found to improve crystallizability. For example, the simulations revealed a redesigning of a water mediated electrostatic interaction (“wet contact”) into a water depleted hydrophobic effect (“dry contact”) and the optimization of a weak hydrogen bonding contact towards a strong one. These findings explained the experimentally found improved crystallizability. Our study emphasizes that it is difficult to derive simple rules for engineering crystallizability but that free energy simulations could be a very useful tool for understanding the contribution of crystal contacts for stability and furthermore could help guide protein engineering strategies to enhance crystallization for technical purposes.
Highlights
It is often thought that proteins are trained by evolution not to aggregate or crystallize as it may interfere with their functions [1]: crystallization in living cells leads to disorders or diseases such as eye cataract [2,3], homozygous hemoglobin C disease, causing a form of anaemia [4], or the so-called “Charcot-Lyden crystals”, leading to bronchial asthma [5,6]
For mutant Q207D, further crystallization experiments at increased protein and polyethylene glycol (PEG) concentrations (10 g L−1 LbADH and 100 g L−1 PEG at 20 °C) were conducted. At both tested crystallization conditions, Q207D crystallized significantly slower and yielded a reduced amount of crystals compared to the wild type
The success of the protein engineering can be evaluated in silico by free energy change calculations
Summary
It is often thought that proteins are trained by evolution not to aggregate or crystallize as it may interfere with their functions [1]: crystallization in living cells leads to disorders or diseases such as eye cataract [2,3], homozygous hemoglobin C disease, causing a form of anaemia [4], or the so-called “Charcot-Lyden crystals”, leading to bronchial asthma [5,6]. Evolution led to families of proteins which derive their functions from a crystallized state: Bacillus thuringiensis produces pore-forming crystal (Cry) proteins that are used as insecticides [7]. Long before the first X-ray crystallography structure of a protein was solved by Nobel Prize winning scientists Max Perutz and John Kendrew in the 1950s [8,9], crystallization has been used for purification purposes. Technical protein crystallization is focused on as an attractive, effective purification method compared to conventional, costly chromatographic methods [13]. Protein crystals are well suited as a delivery tool of biopharmaceuticals [14]
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