Membrane proteins are still heavily underrepresented in the protein data bank (PDB) due to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles due to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and/or amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryoelectron microscopy (cryo-EM).

Bioactive apelin peptide forms ranging in length from 12 to 55 amino acids bind to and activate the apelin receptor (AR or APJ), a class A G-protein coupled receptor. Apelin-12, -17, and -36 isoforms, named according to length, with an additional N-terminal cysteine residue allowed for regiospecific and efficient conjugation of pyrene-maleimide. Through steady-state fluorescence spectroscopy, the emission properties of pyrene in aqueous buffer were compared to those of the pyrene-apelin conjugates both without and with zwitterionic or anionic micelles. Pyrene photophysics are consistent with an expected partitioning into the hydrophobic micellar cores, while pyrene-apelin conjugation prevented this partitioning. Apelin, conversely, is expected to preferentially interact with anionic micelles; pyrene-apelin conjugates appear to lose preferential interaction. Finally, Förster resonance energy transfer between pyrene and tryptophan residues in the N-terminal tail and first transmembrane segment (the AR55 construct, comprising residues 1-55 of the AR) was consistent with efficient nonspecific pyrene-apelin conjugate binding to micelles rather than direct, specific apelin-AR55 binding. This approach provides a versatile fluorophore conjugation strategy for apelin, particularly valuable given that even a highly hydrophobic fluorophore is not deleterious to peptide behavior in membrane-mimetic micellar systems. Figure S2) demonstrating Cys-apelin-36 expression, SUMO cleavage and purification. Concentration dependent emission spectra of Py-Cys-apelin-17 in buffer and surfactant micelles ( Figure S3). Graphical Abstract

The pathway of cellular entry of colicins and viral DNA is a fundamental structure problem that is relevant to an understanding the molecular basis of infectious diseases. The cytotoxin colicin E1 uses the outer membrane/transperiplasmic drug export protein, TolC, for its import. TolC consists of a 12 strand OM b-barrel connected to a 12 strand a-helical tunnel that defines a pathway through the peptidoglycan barrier in the periplasm to the cytoplasmic membrane in which the C-terminal domain of colicin E1 inserts and forms a depolarizing ion channel. The nature of the interaction of the colicin with TolC and the mechanism of its translocation are unknown. Previous studies with planar bilayers showed that colicin E1 occludes TolC channels [1], as do certain colicin T-domain peptides [2]. Here, in vivo protection of sensitive E. coli from colicin E1 by a series of N-terminal colicin peptides is used to probe the interaction of the colicin with TolC, with the goal of defining the sites of TolC-colicin interaction and the mechanism of colicin entry. N-terminal segments '1-40','1-81', and '1-100' of the colicin did not provide cytotoxic protection, nor occlude TolC channels. Segments '1-120', '1-140', 1-190', as well as '41-190' and '57-190' protected efficiently in vivo and occluded TolC with high efficiency. Occlusion required a trans-negative electrical potential and was irreversible. Co-elution of the colicin peptides with TolC on a Superdex 200 column was shown for '41-190', but not for '1-81.' In addition to the correlation with protection in vivo from killing by colicin E1, occlusion efficiency also correlated with a basic pI between colicin residues 82 and 140. [1]