Technical Highlight - February 2012
Short description: An analysis of the effects of protein overexpression gives insight into the difficulties of preparing intramembrane proteins for crystallization.
Despite the recent publication of several breakthrough, high-resolution crystal structures of intramembrane proteins (IMPs), the production of IMPs for structural determination remains challenging. Protein toxicity is a frequent roadblock, widely believed to be related to the limited capacity of the host cell to properly fold and accommodate overexpressed IMPs. However, some IMPs can be highly expressed while others are toxic even at low levels; thus, these limitations cannot be generalized.
To understand the basis of overexpressed IMP toxicity, Hunt and colleagues (PSI NYCOMPS) have now extensively analyzed the response of Escherichia coli to the overexpression of six proteins (including four IMPs). Two of the most toxic IMPs, E. coli GlpT and Helicobacter pylori HP1206, caused similar global gene expression changes, and overexpression caused a more elongated morphology compared to control cells. However, cells overexpressing soluble proteins shared most of these changes, although to a lesser extent than those overexpressing IMPs. Surprisingly, cells overexpressing either soluble proteins or IMPs did not present the standard stationary-phase transcriptional response, nor were the heat-shock response and/or envelope-stress systems upregulated, and no major changes were seen in the expression of genes encoding metabolic enzymes. The absence of these expected responses indicates that there is no inherent deterrent to IMP production in E. coli.
Instead of a general barrier, the results suggest that the toxicities due to IMP overexpression may be related to the behavior of the target proteins themselves. This was supported by the finding that the toxicity of E. coli GlpT overexpression was related to its glycerol transport activity and by the discovery of a spontaneous mutation in E. coli MsbA that reduced its toxicity and its transport activity, as well as spontaneous missense mutations in HP1206. Given that the toxicity of a target IMP likely depends on its activity, the authors suggest that using alternative strains that are less susceptible to the effects of a specific protein may result in improved expression of IMPs. They also suggest that selection of activity-reducing mutations during overexpression may be an unappreciated source of heterogeneity impeding IMP crystallization.
F. Gubellini et al. Physiological response to membrane protein overexpression in E. coli.
Mol. Cell. Proteomics. 10, M111.007930 (2011). doi:10.1074/mcp.M111.007930