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The cell is highly crowded with biomacromolecules, and the excluded volume influences processes such as diffusion, folding, conformation, and aggregation or association of proteins and polynucleic acids. In Escherichia coli, the values reported for the total macromolecular content range from 200 to 400 mg/mL. Knowledge of the macromolecular crowding is needed to understand behavior and especially interactions of biomolecules in vivo, be it for drug development, fundamental knowledge, or to support computational efforts to model the living cell. Direct spatiotemporal readout of the crowding would be a powerful asset to unravel the structure of the cytoplasm and the impact of excluded volume on protein function in living cells. Here, we introduce a Förster resonance energy transfer (FRET) sensor for quantification of the macromolecular crowding and apply the sensor in living cells.

Cells are highly crowded with proteins and polynucleotides. Any reaction that depends on the available volume can be affected by macromolecular crowding, but the effects of crowding in cells are complex and difficult to track. Here, we present a set of Fö rster resonance energy transfer (FRET)-based crowding-sensitive probes and investigate the role of the linker design. We investigate the sensors in vitro and in vivo and by molecular dynamics simulations. We find that in vitro all the probes can be compressed by crowding, with a magnitude that increases with the probe size, the crowder concentration, and the crowder size. We capture the role of the linker in a heuristic scaling model, and we find that compression is a function of size of the probe and volume fraction of the crowder. The FRET changes observed in Escherichia coli are more complicated, where FRETincreases and scaling behavior are observed solely with probes that contain the helices in the linker. The probe with the highest sensitivity to crowding in vivo yields the same macromolecular volume fractions as previously obtained from cell dry weight. The collection of new probes provides more detailed readouts on the macromolecular crowding than a single sensor.

The purpose of this study was to investigate the transepithelial transport and cytoprotective effect of Gln-Ile-Gly-Leu-Phe (QIGLF), an ACE-inhibitory peptide derived from egg white ovalbumin, in human intestinal Caco-2 cell monolayers. The results showed that QIGLF could be absorbed intact through Caco-2 cell monolayers with a P value of (9.11 ± 0.19) × 10 cm/s (transport kinetic parameters: K, 32.37 ± 12.59 mM; V, 1.23 ± 0.49 μM/min cm). The transport was not significantly decreased by sodium azide and Gly-Pro, an ATP synthesis inhibitor and a peptide transporter 1 (PepT1) substrate, respectively, suggesting that transport of QIGLF was not energy-dependent and carrier-mediated. In addition, wortmannin, a transcytosis inhibitor, had little effect on the transport, suggesting that endocytosis was not involved in the transport of QIGLF. However, the transport of QIGLF was increased significantly in the presence of cytochalasin D, a tight junction disruptor, suggesting that paracellular transport via tight junctions was the major transport mechanism for intact QIGLF across Caco-2 cell monolayers. Moreover, QIGLF was added to Caco-2 cells followed by addition of HO, and exhibited significant cytoprotective effect in Caco-2 cells against oxidative stress induced by HO.

Knowledge of the ionic strength in cells is required to understand the in vivo biochemistry of the charged biomacromolecules. Here, we present the first sensors to determine the ionic strength in living cells, by designing protein probes based on Förster resonance energy transfer (FRET). These probes allow observation of spatiotemporal changes in the ionic strength on the single-cell level.

Nitrones are key intermediates in organic synthesis and the pharmaceutical industry. The heterogeneous synthesis of nitrones with multifunctional catalysts is extremely attractive but rarely explored. Herein, we report ultrasmall platinum nanoclusters (PtNCs) encapsulated in amine-functionalized Zr metal-organic framework (MOF), UiO-66-NH (Pt@UiO-66-NH ) as a multifunctional catalyst in the one-pot tandem synthesis of nitrones. By virtue of the cooperative interplay among the selective hydrogenation activity provided by the ultrasmall PtNCs and Lewis acidity/basicity/nanoconfinement endowed by UiO-66-NH , Pt@UiO-66-NH exhibits remarkable activity and selectivity, in comparison to Pt/carbon, Pt@UiO-66, and Pd@UiO-66-NH . Pt@UiO-66-NH also outperforms Pt nanoparticles supported on the external surface of the same MOF (Pt/UiO-66-NH ). To our knowledge, this work demonstrates the first examples of one-pot synthesis of nitrones using recyclable multifunctional heterogeneous catalysts.

Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet it remains necessary to identify and prevent potential artifacts in order to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression in both prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster-maturing FRET donors. We developed an improved version of the donor mTurquoise2 that matures faster than the parent protein. Our analysis shows that using equal maturing fluorophores in FRET-based sensors or using constitutive low expression conditions helps to reduce maturation-induced artifacts, without the need of additional noise-inducing spectral corrections. In general, we show that monitoring and controlling the maturation of fluorescent proteins in living cells is important and should be addressed in in vivo applications of genetically encoded FRET sensors.

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