High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

The structural basis of fatty acid elongation by the ELOVL elongases.

Very long chain fatty acids (VLCFAs) are essential building blocks for the synthesis of ceramides and sphingolipids. The first step in the fatty acid elongation cycle is catalyzed by the 3-keto acyl-coenzyme A (CoA) synthases (in mammals, ELOVL elongases). Although ELOVLs are implicated in common diseases, including insulin resistance, hepatic steatosis and Parkinson's, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which comprises an inverted transmembrane barrel surrounding a 35-Å long tunnel containing a covalently attached product analogue. The structure reveals the substrate-binding sites in the narrow tunnel and an active site deep in the membrane. We demonstrate that chain elongation proceeds via an acyl-enzyme intermediate involving the second histidine in the canonical HxxHH motif. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate, such as X-linked adrenoleukodystrophy.

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.

Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein's insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal's DNA-ratchet function.