Production of constrained L-cyclo-tetrapeptides by epimerization-resistant direct aminolysis.

The synthesis of constrained 12-membered rings is notably difficult. The main challenges result from constraints during the linear peptide cyclization. Attempts to overcome constraints through excessive activation frequently cause peptidyl epimerization, while insufficient activation of the C-terminus hampers cyclization and promotes intermolecular oligomer formation.

Messenger RNA in lipid nanoparticles rescues HEK 293 cells from lipid-induced mitochondrial dysfunction as studied by real time pulse chase NMR, RTPC-NMR, spectroscopy.

Analytical tools to study cell physiology are critical for optimizing drug-host interactions. Real time pulse chase NMR spectroscopy, RTPC-NMR, was introduced to monitor the kinetics of metabolite production in HEK 293T cells treated with COVID-19 vaccine-like lipid nanoparticles, LNPs, with and without mRNA. Kinetic flux parameters were resolved for the incorporation of isotopic label into metabolites and clearance of labeled metabolites from the cells.

Microfluidics delivery of DARPP-32 into HeLa cells maintains viability for in-cell NMR spectroscopy.

High-resolution structural studies of proteins and protein complexes in a native eukaryotic environment present a challenge to structural biology. In-cell NMR can characterize atomic resolution structures but requires high concentrations of labeled proteins in intact cells. Most exogenous delivery techniques are limited to specific cell types or are too destructive to preserve cellular physiology.

Active metabolism unmasks functional protein-protein interactions in real time in-cell NMR.

Protein-protein interactions, PPIs, underlie most cellular processes, but many PPIs depend on a particular metabolic state that can only be observed in live, actively metabolizing cells. Real time in-cell NMR spectroscopy, RT-NMR, utilizes a bioreactor to maintain cells in an active metabolic state. Improvement in bioreactor technology maintains ATP levels at >95% for up to 24 hours, enabling protein overexpression and a previously undetected interaction between prokaryotic ubiquitin-like protein, Pup, and mycobacterial proteasomal ATPase, Mpa, to be detected.

Intact ribosomes drive the formation of protein quinary structure.

Transient, site-specific, or so-called quinary, interactions are omnipresent in live cells and modulate protein stability and activity. Quinary intreactions are readily detected by in-cell NMR spectroscopy as severe broadening of the NMR signals. Intact ribosome particles were shown to be necessary for the interactions that give rise to the NMR protein signal broadening observed in cell lysates and sufficient to mimic quinary interactions present in the crowded cytosol.

Improved sensitivity and resolution of in-cell NMR spectra.

In-cell NMR spectroscopy is a powerful tool to study protein structures and interactions under near physiological conditions in both prokaryotic and eukaryotic living cells. The low sensitivity and resolution of in-cell NMR spectra and limited lifetime of cells over the course of an in-cell experiment have presented major hurdles to wide acceptance of the technique, limiting it to a few select systems. These issues are addressed by introducing the use of the CRINEPT pulse sequence to increase the sensitivity and resolution of in-cell NMR spectra and the use of a bioreactor to maintain cell viability for up to 24h.

The Inescapable Effects of Ribosomes on In-Cell NMR Spectroscopy and the Implications for Regulation of Biological Activity.

The effects of RNA on in-cell NMR spectroscopy and ribosomes on the kinetic activity of several metabolic enzymes are reviewed. Quinary interactions between labelled target proteins and RNA broaden in-cell NMR spectra yielding apparent megadalton molecular weights in-cell. The in-cell spectra can be resolved by using cross relaxation-induced polarization transfer (CRINEPT), heteronuclear multiple quantum coherence (HMQC), transverse relaxation-optimized, NMR spectroscopy (TROSY).

Coupling of sterically demanding peptides by β-thiolactone-mediated native chemical ligation.

The ligation of sterically demanding peptidyl sites such as those involving Val-Val and Val-Pro linkages has proven to be extremely challenging with conventional NCL methods that rely on exogenous thiol additives. Herein, we report an efficient β-thiolactone-mediated additive-free NCL protocol that enables the establishment of these connections in good yield. The rapid NCL was followed by desulfurization.

Interaction proteomics by using in-cell NMR spectroscopy.

A synopsis of in-cell NMR spectroscopic approaches to study interaction proteomics in prokaryotic and eukaryotic cells is presented. We describe the use of in-cell NMR spectroscopy to resolve high resolution protein structures, discuss methodologies for determining and analyzing high and low affinity protein-target structural interactions, including intrinsically disordered proteins, and detail important functional interactions that result from these interactions. SIGNIFICANCE: The ultimate goal of structural and biochemical research is to understand how macromolecular interactions give rise to and regulate biological activity in living cells.

Real-Time In-Cell Nuclear Magnetic Resonance: Ribosome-Targeted Antibiotics Modulate Quinary Protein Interactions.

How ribosome antibiotics affect a wide range of biochemical pathways is not well understood; changes in RNA-mediated protein quinary interactions and consequent activity inside the crowded cytosol may provide one possible mechanism. We developed real-time (RT) in-cell nuclear magnetic resonance (NMR) spectroscopy to monitor temporal changes in protein quinary structure, for ≥24 h, in response to external and internal stimuli. RT in-cell NMR consists of a bioreactor containing gel-encapsulated cells inside a 5 mm NMR tube, a gravity siphon for continuous exchange of medium, and a horizontal drip irrigation system to supply nutrients to the cells during the experiment.

Full Sequence Amino Acid Scanning of θ-Defensin RTD-1 Yields a Potent Anthrax Lethal Factor Protease Inhibitor.

θ-Defensin RTD-1 is a noncompetitive inhibitor of anthrax lethal factor (LF) protease (IC = 390 ± 20 nM, K = 365 ± 20 nM) and a weak inhibitor of other mammalian metalloproteases such as TNFα converting enzyme (TACE) (K = 4.45 ± 0.48 μM).

Efficient recombinant expression of SFTI-1 in bacterial cells using intein-mediated protein trans-splicing.

We report for the first time the recombinant expression of bioactive wild-type sunflower trypsin inhibitor 1 (SFTI-1) inside E. coli cells by making use of intracellular protein trans-splicing in combination with a high efficient split-intein. SFTI-1 is a small backbone-cyclized polypeptide with a single disulfide bridge and potent trypsin inhibitory activity.