The critical importance of conditions: Reconciling GPCR functionality and biophysical findings.

G-protein-coupled receptor (GPCR) activation relies on conformational sampling, a nuanced but functionally key behavior well suited to elucidation by nuclear magnetic resonance (NMR) spectroscopy. In this issue of Structure, Thakur et al.1 demonstrate that judicious choice of experimental conditions for 19F NMR studies of a GPCR enables rationalization of functional and pharmacological behavior, leading to testable hypotheses correlating structure, dynamics, and function.

A recombinant chimeric spider pyriform-aciniform silk with highly tunable mechanical performance.

Spider silks are natural protein-based biomaterials which are renowned for their mechanical properties and hold great promise for applications ranging from high-performance textiles to regenerative medicine. While some spiders can produce several different types of silks, most spider silk types - including pyriform and aciniform silks - are relatively unstudied. Pyriform and aciniform silks have distinct mechanical behavior and physicochemical properties, with materials produced using combinations of these silks currently unexplored. Here, we introduce an engineered chimeric fusion protein consisting of two repeat units of pyriform (Py) silk followed by two repeat units of aciniform (W) silk named Py2W2. This recombinant ∼86.5 kDa protein is amenable to expression and purification from Escherichia coli and exhibits high α-helicity in a fluorinated acid- and alcohol-based solution used to form a dope for wet-spinning. Wet-spinning enables continuous fiber production and post-spin stretching of the wet-spun fibers in air or following submersion in water or ethanol leads to increases in optical anisotropy, consistent with increased molecular alignment along the fiber axis. Mechanical properties of the fibers vary as a function of post-spin stretching condition, with the highest extensibility and strength observed in air-stretched and ethanol-treated fibers, respectively, with mechanics being superior to fibers spun from either constituent protein alone. Notably, the maximum extensibility obtained (∼157 ± 38 %) is of the same magnitude reported for natural flagelliform silks, the class of spider silk most associated with being stretchable. Interestingly, Py2W2 is also water-compatible, unlike its constituent Py2. Fiber-state secondary structure correlates well with the observed mechanical properties, with depleted α-helicity and increased ß-sheet content in cases of increased strength. Py2W2 fibers thus provide enhanced materials behavior in terms of their mechanics, tunability, and fiber properties, providing new directions for design and development of biomaterials suitable and tunable for disparate applications.

Secondary Structure Stabilization of Macrocyclic Antimicrobial Peptides via Cross-Link Swapping.

Lactam cross-links have been employed to stabilize the helical secondary structure and enhance the activity and physiological stability of antimicrobial peptides; however, stabilization of ß-sheets via lactamization has not been observed. In the present study, lactams between the side chains of C- and N-terminal residues have been used to stabilize the ß-sheet conformation in a short ten-residue analogue of chicken angiogenin-4. Designed using a combination of molecular dynamics simulations and Markov state models, the lactam cross-linked peptides are shown to adopt stabilized ß-sheet conformations consistent with simulated structures. Replacement of the peptide side-chain Cys-Cys disulfide by a lactam cross-link enhanced the broad-spectrum antibacterial activity compared to the parent peptide and exhibited greater propensity to induce proinflammatory activity in macrophages. The combination of molecular simulations and conformational and biological analyses of the synthetic peptides provides a useful paradigm for the rational design of therapeutically active peptides with constrained ß-sheet structures.

Localized apelin-17 analogue-bicelle interactions as a facilitator of membrane-catalyzed receptor recognition and binding.

The apelinergic system encompasses two peptide ligand families, apelin and apela, along with the apelin receptor (AR or APJ), a class A G-protein-coupled receptor. This system has diverse physiological effects, including modulating heart contraction, vasodilation/constriction, glucose regulation, and vascular development, with involvement in a variety of pathological conditions. Apelin peptides have been previously shown to interact with and become structured upon binding to anionic micelles, consistent with a membrane-catalyzed mechanism of ligand-receptor binding. To overcome the challenges of observing nuclear magnetic resonance (NMR) spectroscopy signals of a dilute peptide in biological environments, 19F NMR spectroscopy, including diffusion ordered spectroscopy (DOSY) and saturation transfer difference (STD) experiments, was used herein to explore the membrane-interactive behaviour of apelin. NMR-optimized apelin-17 analogues with 4-trifluoromethyl-phenylalanine at various positions were designed and tested for bioactivity through ERK activation in stably-AR transfected HEK 293 T cells. Far-UV circular dichroism (CD) spectropolarimetry and 19F NMR spectroscopy were used to compare the membrane interactions of these analogues with unlabelled apelin-17 in both zwitterionic/neutral and net-negative bicelle conditions. Each analogue binds to bicelles with relatively weak affinity (i.e., in fast exchange on the NMR timescale), with preferential interactions observed at the cationic residue-rich N-terminal and mid-length regions of the peptide leaving the C-terminal end unencumbered for receptor recognition, enabling a membrane-anchored fly-casting mechanism of peptide search for the receptor. In all, this study provides further insight into the membrane-interactive behaviour of an important bioactive peptide, demonstrating interactions and biophysical behaviour that cannot be neglected in therapeutic design.

Elucidating the Role of Optical Activity of Polymers in Protein-Polymer Interactions.

Proteins are biomolecules with potential applications in agriculture, food sciences, pharmaceutics, biotechnology, and drug delivery. Interactions of hydrophilic and biocompatible polymers with proteins may impart proteolytic stability, improving the therapeutic effects of biomolecules and also acting as excipients for the prolonged storage of proteins under harsh conditions. The interactions of hydrophilic and stealth polymers such as poly(ethylene glycol), poly(trehalose), and zwitterionic polymers with various proteins are well studied. This study evaluates the molecular interactions of hydrophilic and optically active poly(vitamin B5 analogous methacrylamide) (poly(B5AMA)) with model proteins by fluorescence spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and circular dichroism (CD) spectroscopy analysis. The optically active hydrophilic polymers prepared using chiral monomers of R-(+)- and S-(-)-B5AMA by the photo-iniferter reversible addition fragmentation chain transfer (RAFT) polymerization showed concentration-dependent weak interactions of the polymers with bovine serum albumin and lysozyme proteins. Poly(B5AMA) also exhibited a concentration-dependent protein stabilizing effect at elevated temperatures, and no effect of the stereoisomers of polymers on protein thermal stability was observed. NMR analysis, however, showed poly(B5AMA) stereoisomer-dependent changes in the secondary structure of proteins.