Spiroligomer-Based Macrocycles for Atomically Precise Membranes.

Here, we report a new class of peptidomimetic macrocycles with well-defined three-dimensional structures and low conformational flexibility. They are assembled from fused-ring spiro-ladder oligomers (spiroligomers) by modular solid-phase synthesis. Two-dimensional nuclear magnetic resonance confirms their shape persistency. Triangular macrocycles of tunable sizes assemble into membranes with atomically precise pores, which exhibit size and shape-dependent molecular sieving towards a series of structurally similar compounds. The exceptional structural diversity and stability of spiroligomer-based macrocycles will be explored for more applications.

Proximity labeling for investigating protein-protein interactions.

The study of protein complexes and protein-protein interactions is of great importance due to their fundamental roles in cellular function. Proximity labeling, often coupled with mass spectrometry, has become a powerful and versatile tool for studying protein-protein interactions by enriching and identifying proteins in the vicinity of a specified protein-of-interest. Here, we describe and compare traditional approaches to investigate protein-protein interactions to current day state-of-the-art proximity labeling methods. We focus on the wide array of proximity labeling strategies and underscore studies using diverse model systems to address numerous biological questions. In addition, we highlight current advances in mass spectrometry-based technology that exhibit promise in improving the depth and breadth of the data acquired in proximity labeling experiments. In all, we show the diversity of proximity labeling strategies and emphasize the broad range of applications and biological inquiries that can be addressed using this technology.

Mapping Angiotensin II Type 1 Receptor-Biased Signaling Using Proximity Labeling and Proteomics Identifies Diverse Actions of Biased Agonists.

Angiotensin II type 1 receptors (AT1Rs) are one of the most widely studied G-protein-coupled receptors. To fully appreciate the diversity in cellular signaling profiles activated by AT1R transducer-biased ligands, we utilized peroxidase-catalyzed proximity labeling to capture proteins in close proximity to AT1Rs in response to six different ligands: angiotensin II (full agonist), S1I8 (partial agonist), TRV055 and TRV056 (G-protein-biased agonists), and TRV026 and TRV027 (ß-arrestin-biased agonists) at 90 s, 10 min, and 60 min after stimulation (ProteomeXchange Identifier PXD023814). We systematically analyzed the kinetics of AT1R trafficking and determined that distinct ligands lead AT1R to different cellular compartments for downstream signaling activation and receptor degradation/recycling. Distinct proximity labeling of proteins from a number of functional classes, including GTPases, adaptor proteins, and kinases, was activated by different ligands suggesting unique signaling and physiological roles of the AT1R. Ligands within the same class, that is, either G-protein-biased or ß-arrestin-biased, shared high similarity in their labeling profiles. A comparison between ligand classes revealed distinct signaling activation such as greater labeling by G-protein-biased ligands on ESCRT-0 complex proteins that act as the sorting machinery for ubiquitinated proteins. Our study provides a comprehensive analysis of AT1R receptor-trafficking kinetics and signaling activation profiles induced by distinct classes of ligands.

Determination of internal energy distributions of laser electrospray mass spectrometry using thermometer ions and other biomolecules.

The internal energy distributions for dried and liquid samples that were vaporized with femtosecond duration laser pulses centered at 800 nm and postionized by electrospray ionization-mass spectrometry (LEMS) were measured and compared with conventional electrospray ionization mass spectrometry (ESI-MS). The internal energies of the mass spectral techniques were determined by plotting the ratio of the intact parent molecular features to all integrated ion intensities of the fragments as a function of collisional energy using benzylpyridinium salts and peptides. Measurements of dried p-substituted benzylpyridinium salts using LEMS resulted in a greater extent of fragmentation in addition to the benzyl cation. The mean relative internal energies, were determined to be 1.62 ± 0.06, 2.0 ± 0.5, and 1.6 ± 0.3 eV for ESI-MS, dried LEMS, and liquid LEMS studies, respectively. Two-photon resonances with the laser pulses likely caused lower survival yields in LEMS analyses of dried samples but not liquid samples. In studies with larger biomolecules, LEMS analyses of dried samples from glass showed a decrease in survival yield compared with conventional ESI-MS for leucine enkephalin and bradykinin of ~15% and 11%, respectively. The survival yields for liquid LEMS analyses were comparable to or better than ESI-MS for benzylpyridinium salts and large biomolecules.

Solid phase synthesis of a functionalized bis-peptide using "safety catch" methodology.

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides(1). Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides(2), which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield(1), but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation. Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an α-amine)(3,4). Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.