Technical
protein crystallization is an alternative to preparative
chromatography for purification of proteins. However, only a few proteins
are satisfactorily crystallizable for this technical purpose. In the
present work, the crystallizability of Lactobacillus
brevis alcohol dehydrogenase (LbADH)
was significantly improved by rational engineering of its crystal
contact patches. The concept was to exchange amino acids at the crystal
contact patches with the objective of (i) surface entropy reduction
(SER) and (ii) enhancement of ionic interactions.
We present three newly designed, enzymatically active LbADH mutants with improved crystallizability: K32A (via SER) and Q126H and Q126K (both via enhancement of ionic interactions).
The wild type crystallized with a low crystallization success rate
in microbatch experiments. All mutants crystallized consistently with
enhanced crystallization kinetics under identical conditions. Mutant
K32A crystallized at reduced protein concentrations. Mutant Q126H
crystallized at reduced concentrations of protein and the crystallization
agent polyethylene glycol. Furthermore, the X-ray structure of mutant
K32A reveals evidence of crystal contact enforcement which does explain
enhanced crystallizability on the atomic level. The increased space–time
yield of mutant K32A in stirred tank crystallizers demonstrates that
rational crystal contact engineering is a powerful tool to promote
technical protein crystallization.
Triose phosphates (TPs) are the primary products of photosynthetic CO2 fixation in chloroplasts, which need to be exported into the cytosol across the chloroplast inner envelope (IE) and outer envelope (OE) membranes to sustain plant growth. While transport across the IE is well understood, the mode of action of the transporters in the OE remains unclear. Here we present the high-resolution nuclear magnetic resonance (NMR) structure of the outer envelope protein 21 (OEP21) from garden pea, the main exit pore for TPs in C3 plants. OEP21 is a cone-shaped β-barrel pore with a highly positively charged interior that enables binding and translocation of negatively charged metabolites in a competitive manner, up to a size of ~1 kDa. ATP stabilizes the channel and keeps it in an open state. Despite the broad substrate selectivity of OEP21, these results suggest that control of metabolite transport across the OE might be possible.
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