Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases

Brigitte Walla,Daniel Bischoff,Dirk Weuster-Botz,Robert Janowski,Dierk Niessing,Nikolas Von Den Eichen

doi:10.3390/cryst11080975

Brigitte Walla, Daniel Bischoff + Show 4 more

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https://doi.org/10.3390/cryst11080975

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Abstract

Protein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational crystal contact engineering. Two selected rational crystal contact mutations, Q126K and T102E, were transferred from the alcohol dehydrogenases of Lactobacillus brevis (LbADH) to Lactobacillus kefir (LkADH). Proteins were expressed in E. coli and batch protein crystallization was performed in stirred crystallizers. Highly similar crystal packing of LkADH wild type compared to LbADH, which is necessary for the transfer of crystal contact engineering strategies, was achieved by aligning purification tag and crystallization conditions, as shown by X-ray diffraction. After comparing the crystal sizes after crystallization of LkADH mutants with the wild type, the mean protein crystal size of LkADH mutants was reduced by 40–70% in length with a concomitant increase in the total amount of crystals (higher number of nucleation events). Applying this measure to the LkADH variants studied results in an order of crystallizability T102E > Q126K > LkADH wild type, which corresponds to the results with LbADH mutants and shows, for the first time, the successful transfer of crystal contact engineering strategies.

Highlights

The enzyme Lactobacillus kefir alcohol dehydrogenase (LkADH) was selected for the transfer of a successful crystal contact engineering strategy established with Lactobacillus brevis alcohol dehydrogenase (LbADH)
Since the LkADH variant described in the literature (PDB identification code (ID): 4RF4, [28]) does not form the same crystal contacts as LbADH, the sequence and crystallization conditions of
Protein production and IMAC purification of His6 _GSG_LkADH (LkADH wild type) was implemented, resulting in purified and active LkADH wild type protein (SDS-PAGE and enzymatic activity are shown in Supplementary Figures S2 and S3)

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Protein crystallization is mainly used for crystallography to obtain the structural information of biological macromolecules. Growing well-diffracting protein crystals for X-ray analysis is still challenging. There are numerous commercially available high-throughput screening kits that can be used to vary extrinsic conditions like pH, buffer type, ionic strength, or precipitant type for receiving protein crystals [1]

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