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. Changes in the characteristic times for alanine production implicated mitochondrial dysfunction as a consequence of treating the cells with lipid nanoparticles, LNPs. Mitochondrial dysfunction was largely abated by inclusion of mRNA in the LNPs, the presence of which increased the size and uniformity of the LNPs. The methodology is applicable to all cultured 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. The feasibility of microfluidics transfection or volume exchange for convective transfer, VECT, as a means to deliver labeled target proteins to HeLa cells for in-cell NMR experiments is demonstrated. VECT delivery does not require optimization or impede cell viability; cells are immediately available for long-term eukaryotic in-cell NMR experiments. In-cell NMR-based drug screening using VECT was demonstrated by collecting spectra of the sensor molecule DARPP32, in response to exogenous administration of Forskolin.

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. Singular value decomposition, SVD, of the NMR spectra collected over the course of Mpa overexpression easily identified the PPIs despite the large variation in background signals due to the highly active metabolome.

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. Recovery of target protein NMR spectra that were broadened in lysates, in vitro and in the presence of purified ribosomes was achieved by RNase A digestion only after the structure of the ribosome was destabilized by removing magnesium ions from the system. Identifying intact ribosomal particles as the major protein-binding component of quinary interactions and consequent spectral peak broadening will facilitate quantitative characterization of macromolecular crowding effects in live cells and streamline models of metabolic activity.

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. Application of advanced pulse sequences and bioreactor during in-cell NMR experiments will facilitate the exploration of a wide range of biological processes.

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). The effect is reproduced in vitro by using reconstituted total cellular RNA and purified ribosome preparations. Furthermore, ribosomal binding antibiotics alter protein quinary structure through protein-ribosome and protein-mRNA-ribosome interactions. The quinary interactions of Adenylate kinase, Thymidylate synthase and Dihydrofolate reductase alter kinetic properties of the enzymes. The results demonstrate that ribosomes may specifically contribute to the regulation of biological activity.

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. The challenge is formidable due to the complexity that arises not only from the number of proteins (genes) expressed by the organism, but also from the combinatorial interactions between them. Despite ongoing efforts to decipher the complex nature of protein interactions, new methods for structurally characterizing protein complexes are needed to fully understand molecular networks. With the onset of in-cell NMR spectroscopy, molecular structures and interactions can be studied under physiological conditions shedding light on the structural underpinning of biological activity.

Coupling of sterically demanding peptides by ß-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 ß-thiolactone-mediated additive-free NCL protocol that enables the establishment of these connections in good yield. The rapid NCL was followed by in situ desulfurization. Reaction rates between ß-thiolactones and conventional thioesters towards NCL were also investigated, and direct aminolysis was ruled out as a possible pathway. Finally, the potent cytotoxic cyclic-peptide axinastatin 1 has been prepared using the developed methodology.

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. We showed that adding antibiotics that bind to the small ribosomal subunit results in more extensive quinary interactions between thioredoxin and mRNA. The results substantiate the idea that RNA-mediated modulation of quinary protein interactions may provide the physical basis for ribosome inhibition and other regulatory pathways.

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 (IC50 = 390 ± 20 nM, Ki = 365 ± 20 nM) and a weak inhibitor of other mammalian metalloproteases such as TNFα converting enzyme (TACE) (Ki = 4.45 ± 0.48 µM). Using full sequence amino acid scanning in combination with a highly efficient "one-pot" cyclization-folding approach, we obtained an RTD-1-based peptide that was around 10 times more active than wild-type RTD-1 in inhibiting LF protease (IC50 = 43 ± 3 nM, Ki = 18 ± 1 nM). The most active peptide was completely symmetrical, rich in Arg and Trp residues, and able to adopt a native RTD-1-like structure. These results show the power of optimized chemical peptide synthesis approaches for the efficient production of libraries of disulfide-rich backbone-cyclized peptides to quickly perform structure-activity relationship studies for optimizing protease inhibitors.

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. Recombinantly produced SFTI-1 was fully characterized by NMR and was observed to actively inhibit trypsin. The in-cell expression of SFTI-1 was very efficient reaching intracellular concentration ≈ 40 µM. This study clearly demonstrates the possibility of generating genetically encoded SFTI-based peptide libraries in live E. coli cells, and is a critical first step for developing in-cell screening and directed evolution technologies using the cyclic peptide SFTI-1 as a molecular scaffold. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 818-824, 2016.