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Challenges in the production of integral membrane proteins for structural studies include low expression levels, incorrect membrane insertion, aggregation and instability. In this report, we describe a “funnel approach” to overcoming these difficulties and demonstrate its efficacy in a case study of 36 prokaryotic P-type transporters. A diverse ensemble of modified constructs is generated and tested for expression in Escherichia coli, membrane localization, detergent extraction, and homogeneity. High-throughput methodologies are implemented throughout the process to facilitate identification of promising targets. We find that the choice of promoter, the choice of source organism providing the cloned gene, and, most importantly, the position of the affinity tag have a great effect on successful production. The latter had pronounced effects at all tested levels, from expression levels observed in whole cells to the extent of membrane insertion, and even on protein function. Following the initial streamlined screening, we were able to fine-tune and produce 9 of the 36 targets as materials suitable for crystallization or other structural studies.

The high-affinity uptake of methionine by Escherichia coli is mediated by MetNI, a member of the methionine uptake transporter family of ATP-binding cassette (ABC) transporters. We previously reported the crystal structure of MetNI at 3.7 A resolution and a brief analysis of the methionine mediated trans-inhibition of the transporters' ATPase activity1. Here, we report two new crystal structures of the MetNI transporter, solved at 2.8 and 4.0 A resolution. While both structures of MetNI reveal the transporter adopting an inward-facing conformation, significant changes are observed in the conformation of the MetI transmembrane, MetN nucleotide binding, and MetN-C2 carboxyl-terminal regulatory domains. The conformational changes can be described primarily as rigid-body movements which result in a partial closing of the MetN nucleotide binding domains, and a simultaneous rotational rearrangement of the MetN-C2 regulatory domain. The kinetic properties of trans-inhibition have also been characterized by an analysis of ligand binding on the ATPase activity of wild type and specific MetN-C2 regulatory domain mutants.1. Kadaba N, Kaiser J, Johnson E, Lee A, and Rees D C. The high affinity E. coli methionine ABC transporter: structure and allosteric regulation. Science. 2008 Jul 11;321(5886):250-3.