A systematic approach to quantitative Western blot analysis.

Advances over the past 25 years have revealed much about how the structural properties of membranes and associated proteins are linked to the thermodynamics and kinetics of membrane protein (MP) folding. At the same time biochemical progress has outlined how cellular proteostasis networks mediate MP folding and manage misfolding in the cell. When combined with results from genomic sequencing, these studies have established paradigms for how MP folding and misfolding are linked to the molecular etiologies of a variety of diseases. This emerging framework has paved the way for the development of a new class of small molecule "pharmacological chaperones" that bind to and stabilize misfolded MP variants, some of which are now in clinical use. In this review, we comprehensively outline current perspectives on the folding and misfolding of integral MPs as well as the mechanisms of cellular MP quality control. Based on these perspectives, we highlight new opportunities for innovations that bridge our molecular understanding of the energetics of MP folding with the nuanced complexity of biological systems. Given the many linkages between MP misfolding and human disease, we also examine some of the exciting opportunities to leverage these advances to address emerging challenges in the development of therapeutics and precision medicine.

Folding and Misfolding of Human Membrane Proteins in Health and Disease: From Single Molecules to Cellular Proteostasis

Plant lipids: Key players of plasma membrane organization and function.

Apelin and apela (ELABELA/ELA/Toddler) are two peptide ligands for a class A G-protein-coupled receptor named the apelin receptor (AR/APJ/APLNR). Ligand-AR interactions have been implicated in regulation of the adipoinsular axis, cardiovascular system, and central nervous system alongside pathological processes. Each ligand may be processed into a variety of bioactive isoforms endogenously, with apelin ranging from 13 to 55 amino acids and apela from 11 to 32, typically being cleaved C-terminal to dibasic proprotein convertase cleavage sites. The C-terminal region of the respective precursor protein is retained and is responsible for receptor binding and subsequent activation. Interestingly, both apelin and apela exhibit isoform-dependent variability in potency and efficacy under various physiological and pathological conditions, but most studies focus on a single isoform. Biophysical behavior and structural properties of apelin and apela isoforms show strong correlations with functional studies, with key motifs now well determined for apelin. Unlike its ligands, the AR has been relatively difficult to characterize by biophysical techniques, with most characterization to date being focused on effects of mutagenesis. This situation may improve following a recently reported AR crystal structure, but there are still barriers to overcome in terms of comprehensive biophysical study. In this review, we summarize the three components of the apelinergic system in terms of structure-function correlation, with a particular focus on isoform-dependent properties, underlining the potential for regulation of the system through multiple endogenous ligands and isoforms, isoform-dependent pharmacological properties, and biological membrane-mediated receptor interaction. © 2018 American Physiological Society. Compr Physiol 8:407-450, 2018.

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Apelinergic System Structure and Function

The expression and integration of membrane proteins into vesicle membranes is a critical step in the design of cell-mimetic biosensors, bioreactors, and artificial cells. While membrane proteins have been integrated into a variety of nonnatural membranes, the effects of the chemical and physical properties of these vesicle membranes on protein behavior remain largely unknown. Nonnatural amphiphiles, such as diblock copolymers, provide an interface that can be synthetically controlled to better investigate this relationship. Here, we focus on the initial step in a membrane protein’s life cycle: expression and folding. We observe improvements in both the folding and overall production of a model mechanosensitive channel protein, the mechanosensitive channel of large conductance, during cell-free reactions when vesicles containing diblock copolymers are present. By systematically tuning the membrane composition of vesicles through incorporation of a poly(ethylene oxide)-b-poly(butadiene) diblock copolymer, we show that membrane protein folding and production can be improved over that observed in traditional lipid vesicles. We then reproduce this effect with an alternate membrane-elasticizing molecule, C12E8. Our results suggest that global membrane physical properties, specifically available membrane surface area and the membrane area expansion modulus, significantly influence the folding and yield of a membrane protein. Furthermore, our results set the stage for explorations into how nonnatural membrane amphiphiles can be used to both study and enhance the production of biological membrane proteins.

https://www.pnas.org/content/pnas/116/10/4031.full.pdf

Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing 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, owing 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 the 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 cryo-electron microscopy (cryo-EM).

Current strategies for protein production and purification enabling membrane protein structural biology.

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, Forster resonance energy transfer between pyrene and tryptophan residues in the N-terminal tail and first transmembrane segment (the AR55 con...

/pdf/pyrene-apelin-conjugation-modulates-fluorophore-and-peptide-4klahkhe2o.pdf

Robin E. Patterson

Papers

Current strategies for protein production and purification enabling membrane protein structural biology.

Pyrene-Apelin Conjugation Modulates Fluorophore- and Peptide-Micelle Interactions.