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Intracellular macromolecular crowding impacts biomacromolecule behavior, including oligomerization, phase separation, and diffusion. However, understanding crowding effects in cells is challenging as cells respond and adapt to perturbations. Therefore, replicating in-cell crowding in liposomes would provide a good alternative to studying the consequences of macromolecular crowding. Here, we achieve physiological macromolecular crowding levels using Escherichia coli lysates in liposomes, as verified with a macromolecular crowding sensor. We shrink liposomes with a gradient-wise osmotic upshift to reach the high macromolecular crowding effects. We see that lysate induces higher macromolecular crowding than BSA at the same mg/mL, showing the need to use lysates to replicate in-cell behavior. We study the consequences of small cosolutes on macromolecular crowding and see that sugars and ATP modulate the lysate macromolecular crowding, implying they would also affect macromolecular crowding in cells. These artificial cells display the same crowding as E. coli at 220-300 mg/mL lysate and the same crowding as HEK293T at 50-100 mg/mL lysate. Hence, these artificial cells are a platform for obtaining information on physiologically relevant macromolecular crowding effects in a controlled environment.

Recently, we have investigated the sensitivity of an mEGFP-linker-mScarlet-I construct (GE2.3) in response to macromolecular crowding using ensemble time-resolved two-photon (2P) fluorescence measurements [Mersch et al. , Phys. Chem. Chem. Phys. 2024, 26 (5), 3927-3940] as a point of reference for developing a single-molecule approach for Förster resonance energy transfer (FRET). Here, we investigate the fluorescence fluctuations, FRET, molecular brightness, and translational diffusion of GE2.3 as a model system using fluorescence correlation spectroscopy (FCS), at the single molecule level, as a function of the excitation and detection wavelengths of the donor (mEGFP) and the acceptor (mScarlet-I). We hypothesize that the molecular brightness (number of fluorescence photons per molecule) of the donor of GE2.3, in the presence and absence of the acceptor, would be distinct due to FRET at the single-molecule level. To test this hypothesis, we used wavelength-dependent FCS to quantify the molecular brightness of intact and enzymatically cleaved GE2.3 as a function of Ficoll-70 (a crowding agent, 0-300 g L -1 ) at room temperature. Our results indicate that the molecular brightness of intact GE2.3 in a buffer is smaller than that of the cleaved counterpart under 488-nm excitation of the donor, which is attributed to FRET. In contrast, the molecular brightness of both cleaved and intact GE2.3 seems to be the same under the 561-nm excitation of the acceptor due to the absence of FRET. Our results also show that the FRET efficiency of GE2.3 increases as the concentration of Ficoll increases up to 200 g L -1 , which agrees with our previous time-resolved 2P-fluorescence measurements. Fluctuation autocorrelation analysis shows that the translational diffusion of intact and cleaved GE2.3 sensors deviates from the Stokes-Einstein model in Ficoll crowded solutions. Additionally, we highlight the multiscale translational and rotational diffusion coefficients of GE2.3 in terms of the average distance between neighboring Ficoll molecules, over the same concentration range, to elucidate the spatio-temporal scaling aspect of FRET and protein-protein interactions. These single-molecule studies would be beneficial for future studies in living cells, where very low GE2.3 expression levels will be required as compared with ensemble, time-resolved 2P-fluorescence measurements.

Weak nonspecific interactions between biomacromolecules determine the cytoplasmic organization. Despite their importance, it is challenging to determine these interactions in the intracellular dense and heterogeneous mixture of biomacromolecules. Here, we develop a method to indicate electrostatic and hydrophobic associative interactions and map these interactions. The method relies on a genetically encoded probe containing a sensing peptide and a circularly permuted green fluorescent protein that provides a ratiometric readout. Inside bacterial and mammalian cells, we see that the cytoplasmic components interact strongly with cationic and hydrophobic probes but not with neutral hydrophilic probes, which remain inert. The Escherichia coli cytoplasm interacts strongly with highly negatively charged hydrophilic probes, but the HEK293T cytoplasm does not. These associative interactions are modulated by ATP depletion. Hence, the nonspecific associative interaction profile in cells is condition- and species-dependent.

Competing Interests: Declaration of interests The author declares no competing interests.

Macromolecular crowding affects the activity of proteins and functional macromolecular complexes in all cells, including bacteria. Crowding, together with physicochemical parameters such as pH, ionic strength, and the energy status, influences the structure of the cytoplasm and thereby indirectly macromolecular function. Notably, crowding also promotes the formation of biomolecular condensates by phase separation, initially identified in eukaryotic cells but more recently discovered to play key functions in bacteria. Bacterial cells require a variety of mechanisms to maintain physicochemical homeostasis, in particular in environments with fluctuating conditions, and the formation of biomolecular condensates is emerging as one such mechanism. In this work, we connect physicochemical homeostasis and macromolecular crowding with the formation and function of biomolecular condensates in the bacterial cell and compare the supramolecular structures found in bacteria with those of eukaryotic cells. We focus on the effects of crowding and phase separation on the control of bacterial chromosome replication, segregation, and cell division, and we discuss the contribution of biomolecular condensates to bacterial cell fitness and adaptation to environmental stress.

Macromolecular crowding affects many cellular processes such as diffusion, biochemical reaction kinetics, protein-protein interactions, and protein folding. Mapping the heterogeneous, dynamic crowding in living cells or tissues requires genetically encoded, site-specific, crowding sensors that are compatible with quantitative, noninvasive fluorescence micro-spectroscopy. Here, we carried out time-resolved 2P-fluorescence measurements of a new mEGFP-linker-mScarlet-I macromolecular crowding construct (GE2.3) to characterize its environmental sensitivity in biomimetic crowded solutions (Ficoll-70, 0-300 g L -1 ) via Förster resonance energy transfer (FRET) analysis. The 2P-fluorescence lifetime of the donor (mEGFP) was measured under magic-angle polarization, in the presence (intact) and absence (enzymatically cleaved) of the acceptor (mScarlet - I), as a function of the Ficoll-70 concentration. The FRET efficiency was used to quantify the sensitivity of GE2.3 to macromolecular crowding and to determine the environmental dependence of the mEGFP-mScarlet - I distance. We also carried out time-resolved 2P-fluorescence depolarization anisotropy to examine both macromolecular crowding and linker flexibility effects on GE2.3 rotational dynamics within the context of the Stokes-Einstein model as compared with theoretical predictions based on its molecular weight. These time-resolved 2P-fluorescence depolarization measurements and conformational population analyses of GE2.3 were also used to estimate the free energy gain upon the structural collapse in crowded environment. Our results further the development of a rational engineering design for bioenvironmental sensors without the interference of cellular autofluorescence. Additionally, these results in well-defined environments will inform our future in vivo studies of genetically encoded GE2.3 towards the mapping of the crowded intracellular environment under different physiological conditions.

Cellular ageing described at the molecular level is a multifactorial process that leads to a spectrum of ageing trajectories. There has been recent discussion about whether a decline in physicochemical homeostasis causes aberrant phase transitions, which are a driver of ageing. Indeed, the function of all biological macromolecules, regardless of their participation in biomolecular condensates, depends on parameters such as pH, crowding, and redox state. We expand on the physicochemical homeostasis hypothesis and summarise recent evidence that the intracellular milieu influences molecular processes involved in ageing., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Therapeutic antibodies are the key treatment option for various cytokine-mediated diseases, such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. However, systemic injection of these antibodies can cause side effects and suppress the immune system. Moreover, clearance of therapeutic antibodies from the blood is limiting their efficacy. Here, water-swollen microgels are produced with a size of 25 µm using droplet-based microfluidics. The microgels are functionalized with TNFα antibodies to locally scavenge the pro-inflammatory cytokine TNFα. Homogeneous distribution of TNFα-antibodies is shown throughout the microgel network and demonstrates specific antibody-antigen binding using confocal microscopy and FLIM-FRET measurements. Due to the large internal accessibility of the microgel network, its capacity to bind TNFα is extremely high. At a TNFα concentration of 2.5 µg mL -1 , the microgels are able to scavenge 88% of the cytokine. Cell culture experiments reveal the therapeutic potential of these microgels by protecting HT29 colorectal adenocarcinoma cells from TNFα toxicity and resulting in a significant reduction of COX II and IL8 production of the cells. When the microgels are incubated with stimulated human macrophages, to mimic the in vivo situation of inflammatory bowel disease, the microgels scavenge almost all TNFα that is produced by the cells., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

The intracellular milieu is crowded with biomacromolecules. Macromolecular crowding changes the interactions, diffusion, and conformations of biomacromolecules. Changes in intracellular crowding have been mostly ascribed to differences in biomacromolecule concentration. However, spatial organization of these molecules should play a significant role in crowding effects. Here, we find that cell wall damage causes increased crowding effects in the Escherichia coli cytoplasm. Using a genetically encoded macromolecular crowding sensor, we see that crowding effects in spheroplasts and penicillin-treated cells well surpass crowding effects obtained using hyperosmotic stress. The crowding increase is not because of osmotic pressure, cell shape, or volume changes and therefore not crowder concentration. Instead, a genetically encoded nucleic acid stain and a DNA stain show cytoplasmic mixing and nucleoid expansion, which could cause these increased crowding effects. Our data demonstrate that cell wall damage alters the biochemical organization in the cytoplasm and induces significant conformational changes in a probe protein., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

The intracellular environment is crowded with macromolecules that influence biochemical equilibria and biomacromolecule diffusion. The incorporation of such crowding in synthetic cells would be needed to mimic the biochemistry of living cells. However, only a few methods provide crowded artificial cells, moreover providing cells with either heterogeneous size and composition or containing a significant oil fraction. Therefore, a method that generates monodisperse liposomes with minimal oil content and tunable macromolecular crowding using polydimethylsiloxane (PDMS)-based microfluidics is presented. Lipid stabilized water-in-oil-in-water emulsions that are stable for at least several months and with a high macromolecular crowder concentration that can be controlled with the external osmolality are formed. A crucial feature is that the oil phase can be removed using high flow conditions at any point after production, providing the highly crowded liposomes. Genetically encoded macromolecular crowding sensors show that the high level of macromolecular crowding in the emulsions is fully retained throughout the generation of minimal-oil lipid bilayers. This modular and robust platform will serve the study of biochemistry under physiologically relevant crowding conditions., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Proteins assemble into a variety of dynamic and functional structures. Their structural transitions are often challenging to distinguish inside cells, particularly with a high spatiotemporal resolution. Here, we present a fluorescence resonance energy transfer (FRET)-based method for continuous and high-throughput monitoring of protein self-assemblies to reveal well-resolved transient intermediate states. Intermolecular FRET with both the donor and acceptor proteins at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We apply this method to monitor self-assembly of three proteins. We show that the mutant Huntingtin exon1 (mHttex1) first forms less-ordered assemblies, which develop into fibril-like aggregates, and demonstrate that the chaperone protein DNAJB6b increases the critical saturation concentration of mHttex1. We also monitor the structural changes in fused in sarcoma (FUS) condensates. This method adds to the toolbox for protein self-assembly structure and kinetics determination, and implementation with native or non-native proteins can inform studies involving protein condensation or aggregation., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1-linker-mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kay, Aplin, Simonet, Beenken, Miller, Libal, Boersma, Sheets and Heikal.)

The regulation of enzyme activity is a method to control biological function. We report two systems enabling the ultrasound-induced activation of thrombin, which is vital for secondary hemostasis. First, we designed polyaptamers, which can specifically bind to thrombin, inhibiting its catalytic activity. With ultrasound generating inertial cavitation and therapeutic medical focused ultrasound, the interactions between polyaptamer and enzyme are cleaved, restoring the activity to catalyze the conversion of fibrinogen into fibrin. Second, we used split aptamers conjugated to the surface of gold nanoparticles (AuNPs). In the presence of thrombin, these assemble into an aptamer tertiary structure, induce AuNP aggregation, and deactivate the enzyme. By ultrasonication, the AuNP aggregates reversibly disassemble releasing and activating the enzyme. We envision that this approach will be a blueprint to control the function of other proteins by mechanical stimuli in the sonogenetics field., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Eukaryotic cells exploit dynamic and compartmentalized ionic strength to impact a myriad of biological functions such as enzyme activities, protein-protein interactions, and catalytic functions. Herein, we investigated the fluorescence depolarization dynamics of recently developed ionic strength biosensors (mCerulean3-linker-mCitrine) in Hofmeister salt (KCl, NaCl, NaI, and Na 2 SO 4 ) solutions. The mCerulean3-mCitrine acts as a Förster resonance energy transfer (FRET) pair, tethered together by two oppositely charged α-helices in the linker region. We developed a time-resolved fluorescence depolarization anisotropy approach for FRET analyses, in which the donor (mCerulean3) is excited by 425-nm laser pulses, followed by fluorescence depolarization analysis of the acceptor (mCitrine) in KE (lysine-glutamate), arginine-aspartate, and arginine-glutamate ionic strength sensors with variable amino acid sequences. Similar experiments were carried out on the cleaved sensors as well as an E6G2 construct, which has neutral α-helices in the linker region, as a control. Our results show distinct dynamics of the intact and cleaved sensors. Importantly, the FRET efficiency decreases and the donor-acceptor distance increases as the environmental ionic strength increases. Our chemical equilibrium analyses of the collapsed-to-stretched conformational state transition of KE reveal that the corresponding equilibrium constant and standard Gibbs free energy changes are ionic strength dependent. We also tested the existing theoretical models for FRET analyses using steady-state anisotropy, which reveal that the angle between the dipole moments of the donor and acceptor in the KE sensor are sensitive to the ionic strength. These results help establish the time-resolved depolarization dynamics of these genetically encoded donor-acceptor pairs as a quantitative means for FRET analysis, which complement traditional methods such as time-resolved fluorescence for future in vivo studies., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Ultrasound (US) produces cavitation-induced mechanical forces stretching and breaking polymer chains in solution. This type of polymer mechanochemistry is widely used for synthetic polymers, but not biomacromolecules, even though US is biocompatible and commonly used for medical therapy as well as in vivo imaging. The ability to control protein activity by US would thus be a major stepping-stone for these disciplines. Here, we provide the first examples of selective protein activation and deactivation by means of US. Using GFP as a model system, we engineer US sensitivity into proteins by design. The incorporation of long and highly charged domains enables the efficient transfer of force to the protein structure. We then use this principle to activate the catalytic activity of trypsin by inducing the release of its inhibitor. We expect that this concept to switch "on" and "off" protein activity by US will serve as a blueprint to remotely control other bioactive molecules., (© 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The intracellular environment contains a high concentration of biomacromolecules that present steric barriers and ample surface area for weak chemical interactions. Consequently, these forces influence protein conformations and protein self-assembly, with an outcome that depends on the sum of the effects resulting from crowding. Linkers are disordered domains that lack tertiary structure, and this flexible nature would render them susceptible to compression or extension under crowded conditions, compared to the equilibrium conformation in a dilute buffer. The change in distance between the linked proteins can become essential where it attenuates protein activity. In this chapter, we first discuss the experimental findings in vitro and in the cell on how linkers and other relevant macromolecules are affected by crowding. We focus on the dependence on the linker's size, flexibility, and the intra- and intermolecular interactions. Although the experimental data on the systematic variation of proteins in a buffer and cells is limited, extrapolating the available insights allows us to propose a protocol on how to engineer the directionality of crowding effects in the linker. Finally, we describe a straightforward experimental protocol on the determination of crowding sensitivity in a buffer and cell., (© 2021 Elsevier Inc. All rights reserved.)

Proteins are essential and abundant components of cellular membranes. Being densely packed within the limited surface area, proteins fulfil essential tasks for life, which include transport, signalling and maintenance of cellular homeostasis. The high protein density promotes nonspecific interactions, which affect the dynamics of the membrane-associated processes, but also contribute to higher levels of membrane organization. Here, we provide a comprehensive summary of the most recent findings of diverse effects resulting from high protein densities in both living membranes and reconstituted systems and display why the crowding phenomenon should be considered and assessed when studying cellular pathways. Biochemical, biophysical and computational studies reveal effects of crowding on the translational mobility of proteins and lipids, oligomerization and clustering of integral membrane proteins, and also folding and aggregation of proteins at the lipid membrane interface. The effects of crowding pervade to larger length scales, where interfacial and transmembrane crowding shapes the lipid membrane. Finally, we discuss the design and development of fluorescence-based sensors for macromolecular crowding and the perspectives to use those in application to cellular membranes and suggest some emerging topics in studying crowding at biological interfaces., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Current bacterial RNA switches suffer from lack of versatile inputs and are difficult to engineer. We present versatile and modular RNA switches that are trans-encoded and based on tRNA-mimicking structures (TMSs). These switches provide a high degree of freedom for reengineering and can thus be designed to accept a wide range of inputs, including RNA, small molecules, and proteins. This powerful approach enables control of the translation of protein expression from plasmid and genome DNA., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here, we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells, we observe drastic changes in organellar volume, leading to crowding on the micrometer scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells., Competing Interests: SM, DT, MC, IR, OT, AS, CL, AB, LV No competing interests declared, MK Reviewing editor, eLife, (© 2020, Mouton et al.)

Attachment of lipophilic groups is an important post-translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids, or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine-based trifunctional linker. The linker allows the facile incorporation of two different functionalities at a cysteine residue in a single step. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α-helical model peptide WALP with or without here and in all cases below; palmitoyl groups in giant unilamellar vesicles that contain a liquid-ordered (L o ) and liquid-disordered (L d ) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the L d phase. These data demonstrate that the L o domain in model membranes is highly unfavorable for a single membrane-spanning peptide., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Electrostatic interactions play a vital role in nature. Biomacromolecules such as proteins are orchestrated by electrostatics, among other intermolecular forces, to assemble and organize biochemistry. Natural proteins with a high net charge exist in a folded state or are unstructured and can be an inspiration for scientists to artificially supercharge other protein entities. Recent findings show that supercharging proteins allows for control of their properties such as temperature resistance and catalytic activity. One elegant method to transfer the favorable properties of supercharged proteins to other proteins is the fabrication of fusions. Genetically engineered, supercharged unstructured polypeptides (SUPs) are just one promising fusion tool. SUPs can also be complexed with artificial entities to yield thermotropic and lyotropic liquid crystals and liquids. These architectures represent novel bulk materials that are sensitive to external stimuli. Interestingly, SUPs undergo fluid-fluid phase separation to form coacervates. These coacervates can even be directly generated in living cells or can be combined with dissipative fiber assemblies that induce life-like features. Supercharged proteins and SUPs are developed into exciting classes of materials. Their synthesis, structures, and properties are summarized. Moreover, potential applications are highlighted and challenges are discussed., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Living cells are complex, crowded, and dynamic and continually respond to environmental and intracellular stimuli. They also have heterogeneous ionic strength with compartmentalized variations in both intracellular concentrations and types of ions. These challenges would benefit from the development of quantitative, noninvasive approaches for mapping the heterogeneous ionic strength fluctuations in living cells. Here, we investigated a class of recently developed ionic strength sensors that consists of mCerulean3 (a cyan fluorescent protein) and mCitrine (a yellow fluorescent protein) tethered via a linker made of two charged α-helices and a flexible loop. The two helices are designed to bear opposite charges, which is hypothesized to increase the ionic screening and therefore a larger intermolecular distance. In these protein constructs, mCerulean3 and mCitrine act as a donor-acceptor pair undergoing Förster resonance energy transfer (FRET) that is dependent on both the linker amino acids and the environmental ionic strength. Using time-resolved fluorescence of the donor (mCerulean3), we determined the sensitivity of the energy transfer efficiencies and the donor-acceptor distances of these sensors at variable concentrations of the Hofmeister series of salts (KCl, LiCl, NaCl, NaBr, NaI, Na 2 SO 4 ). As controls, similar measurements were carried out on the FRET-incapable, enzymatically cleaved counterparts of these sensors as well as a construct designed with two electrostatically neutral α-helices (E6G2). Our results show that the energy transfer efficiencies of these sensors are sensitive to both the linker amino acid sequence and the environmental ionic strength, whereas the sensitivity of these sensors to the identity of the dissolved ions of the Hofmeister series of salts seems limited. We also developed a theoretical framework to explain the observed trends as a function of the ionic strength in terms of the Debye screening of the electrostatic interaction between the two charged α-helices in the linker region. These controlled solution studies represent an important step toward the development of rationally designed FRET-based environmental sensors while offering different models for calculating the energy transfer efficiency using time-resolved fluorescence that is compatible with future in vivo studies.

Genetically encoded Förster resonance energy transfer (FRET)-based probes allow a sensitive readout for different or specific parameters in the living cell. We previously demonstrated how FRET-based probes could quantify macromolecular crowding with high spatio-temporal resolution and under various conditions. Here, we present a protocol developed for the use of FRET-based crowding probes in baker's yeast, but the general considerations also apply to other species, as well as other FRET-based sensors. This method allows straightforward detection of macromolecular crowding under challenging conditions often presented by living cells.

Escherichia coli adapts to changing environmental osmolality to survive and maintain growth. It has been shown that the diffusion of green fluorescent protein (GFP) in cells adapted to osmotic upshifts is higher than expected from the increase in biopolymer volume fraction. To better understand the physicochemical state of the cytoplasm in adapted cells, we now follow the macromolecular crowding during adaptation with fluorescence resonance energy transfer (FRET)-based sensors. We apply an osmotic upshift and find that after an initial increase, the apparent crowding decreases over the course of hours to arrive at a value lower than that before the osmotic upshift. Crowding relates to cell volume until cell division ensues, after which a transition in the biochemical organization occurs. Analysis of single cells by microfluidics shows that changes in cell volume, elongation, and division are most likely not the cause for the transition in organization. We further show that the decrease in apparent crowding upon adaptation is similar to the apparent crowding in energy-depleted cells. Based on our findings in combination with literature data, we suggest that adapted cells have indeed an altered biochemical organization of the cytoplasm, possibly due to different effective particle size distributions and concomitant nanoscale heterogeneity. This could potentially be a general response to accommodate higher biopolymer fractions yet retaining crowding homeostasis, and it could apply to other species or conditions as well. IMPORTANCE Bacteria adapt to ever-changing environmental conditions such as osmotic stress and energy limitation. It is not well understood how biomolecules reorganize themselves inside Escherichia coli under these conditions. An altered biochemical organization would affect macromolecular crowding, which could influence reaction rates and diffusion of macromolecules. In cells adapted to osmotic upshift, protein diffusion is indeed faster than expected on the basis of the biopolymer volume fraction. We now probe the effects of macromolecular crowding in cells adapted to osmotic stress or depleted in metabolic energy with a genetically encoded fluorescence-based probe. We find that the effective macromolecular crowding in adapted and energy-depleted cells is lower than in unstressed cells, indicating major alterations in the biochemical organization of the cytoplasm., (Copyright © 2019 Liu et al.)

DNA is more than a carrier of genetic information: It is a highly versatile structural motif for the assembly of nanostructures, giving rise to a wide range of functionalities. In this regard, the structure programmability is the main advantage of DNA over peptides, proteins, and small molecules. DNA amphiphiles, in which DNA is covalently bound to synthetic hydrophobic moieties, allow interactions of DNA nanostructures with artificial lipid bilayers and cell membranes. These structures have seen rapid growth with great potential for medical applications. In this Review, the current state of the art of the synthesis of DNA amphiphiles and their assembly into nanostructures are first summarized. Next, an overview on the interaction of these DNA amphiphiles with membranes is provided, detailing on the driving forces and the stability of the interaction. Moreover, the interaction with cell surfaces in respect to therapeutics, biological sensing, and cell membrane engineering is highlighted. Finally, the challenges and an outlook on this promising class of DNA hybrid materials are discussed., Competing Interests: The authors declare no conflict of interest.

Living cells are crowded with macromolecules and organelles, which affect a myriad of biochemical processes. As a result, there is a need for sensitive molecular sensors for quantitative, site-specific assessment of macromolecular crowding. Here, we investigated the excited-state dynamics of recently developed hetero-FRET sensors (mCerulean3-linker-mCitrine) in homogeneous and heterogeneous environments using time-resolved fluorescence measurements, which are compatible with fluorescence lifetime imaging microscopy (FLIM). The linker in these FRET constructs, which tether the mCerulean3 (the donor) and mCitrine (the acceptor), vary in both length and flexibility. Glycerol and Ficoll-70 solutions were used for homogeneous and heterogeneous environments, respectively, at variable concentrations. The wavelength-dependent studies suggest that the 425-nm excitation and the 475-nm emission of the donor are best suited for quantitative assessment of the energy transfer efficiency and the donor-acceptor distance of these FRET probes. Under the same experimental conditions, the enzymatically cleaved counterpart of these probes was used as a control as well as a means to account for the changes in the environmental refractive indices. Our results indicate that the energy transfer efficiency of these FRET probes increases as the linker becomes shorter and more flexible in pure buffer at room temperature. In addition, the FRET probes favor a compact structure with enhanced energy transfer efficiency and a shorter donor-acceptor distance in the heterogeneous, polymer-crowded environment due to steric hindrance. In contrast, the stretched conformation of these FRET probes is more favorable in the viscous, homogeneous environment with a reduced energy transfer efficiency and relatively larger donor-acceptor distance as compared with those in pure buffer, which was attributed to a reduced structural fluctuation of the mCerulean3-mCitrine FRET pair in the viscous, more restrictive glycerol-enriched buffer. Our findings will help to advance the potential of these hetero-FRET probes using FLIM for spatio-temporal assessment of the compartmentalized crowding in living cells.

Macromolecular crowding is prevalent in all living cells due to the presence of large biomolecules and organelles. Cellular crowding is heterogeneous and is known to influence biomolecular transport, biochemical reactions, and protein folding. Emerging evidence suggests that some cell pathologies may be correlated with compartmentalized crowding. As a result, there is a need for robust biosensors that are sensitive to crowding as well as quantitative, noninvasive fluorescence methods that are compatible with living cells studies. Here, we have developed a model that describes the rotational dynamics of hetero-Förster resonance energy transfer (FRET) biosensors as a means to determine the energy-transfer efficiency and donor-acceptor distance. The model was tested on wavelength-dependent time-resolved fluorescence anisotropy of hetero-FRET probes (mCerulean3-linker-mCitrine) with variable linkers in both crowded (Ficoll-70) and viscous (glycerol) solutions at room temperature. Our results indicate that the energy-transfer efficiencies of these FRET probes increase as the linker becomes shorter and more flexible in pure buffer at room temperature. In addition, the FRET probes favor compact structures with enhanced energy-transfer efficiencies and a shorter donor-acceptor distance in the heterogeneous, polymer-crowded environment due to steric hindrance. In contrast, the extended conformation of these FRET probes is more favorable in viscous, homogeneous environments with a reduced energy-transfer efficiency compared to those in pure buffer, which we attribute to reduced structural fluctuations of the mCerulean3-mCitrine FRET pair in the glycerol-enriched buffer. Our results represent an important step toward the application of quantitative and noninvasive time-resolved fluorescence anisotropy of hetero-FRET probes to investigate compartmentalized macromolecular crowding and protein-protein interactions in living cells as well as in controlled environments.

That diffusion is important for the proper functioning of cells is without question. The extent to which the diffusion coefficient is important is explored here for prokaryotic cells. We discuss the principles of diffusion focusing on diffusion-limited reactions, summarize the known values for diffusion coefficients in prokaryotes and in in vitro model systems, and explain a number of cases where diffusion coefficients are either limiting for reaction rates or necessary for the existence of phenomena. We suggest a number of areas that need further study including expanding the range of organism growth temperatures, direct measurements of diffusion limitation, expanding the range of cell sizes, diffusion limitation for membrane proteins, and taking into account cellular context when assessing the possibility of diffusion limitation.

Living cells are crowded with dynamic distributions of macromolecules and organelles that influence protein diffusion, molecular transport, biochemical reactions, and protein assembly. Here, we test the hypothesis that the diffusion of single molecules deviates from Brownian motion as described by the Stokes-Einstein model in a manner that depends on the viscosity range, the chemical structure of both the diffusing species and the crowding agents, and the spatio-temporal resolution of the employed analytical methods. Our size-dependent fluorescent probes are rhodamine-110, quantum dots, enhanced green fluorescent proteins (EGFP), and mCerulean3-linker-mCitrine FRET probes with various linker length and flexibility. Using fluorescence correlation spectroscopy (FCS), we investigated the translational diffusion of structure-dependent fluorescent probes, at the single-molecule level, in homogeneous (glycerol) and heterogeneous (Ficoll-70) solutions as a function of the bulk viscosity. Complementary rotational diffusion studies using time-resolved anisotropy enable us to assess weak interactions in crowded and viscous environments. Overall, our results show negative deviation from the Stokes-Einstein model in a fluorophore- and environment-dependent manner. In addition, the deviation between the FCS-measured hydrodynamic radius of the FRET probes in a buffer at room temperature and the molecular-weight based estimate (Perrin equation) as the number of the amino acid residues in the linker increases. These studies are essential for quantitative biophysics using fluorescence- and diffusion-based studies of protein-protein interactions and biomolecular transport in living cells.

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.

Genetically encoded Förster resonance energy transfer (FRET)-based biosensors for the quantification of ligand molecules change the magnitude of FRET between two fluorescent proteins upon binding a target metabolite. When highly sensitive sensors are being designed, extensive sensor optimization is essential. However, it is often difficult to verify the ideas of modifications made to a sensor during the sensor optimization process because of the limited information content of ensemble FRET measurements. In contrast, single-molecule detection provides detailed information and higher accuracy. Here, we investigated a set of glucose and crowding sensors on the single-molecule level. We report the first comprehensive single-molecule study of FRET-based biosensors with reasonable counting statistics and identify characteristics in the single-molecule FRET histograms that constitute fingerprints of sensor performance. Hence, our single-molecule approach extends the toolbox of methods aiming to understand and optimize the design of FRET-based biosensors.

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.

Living cells are crowded with macromolecules and organelles. As a result, there is an urgent need for molecular sensors for quantitative, site-specific assessment of the macromolecular crowding effects on a myriad of biochemical processes toward quantitative cell biology and biophysics. Here we investigate the excited-state dynamics and translational diffusion of a novel FRET sensor (mCerulean-linker-mCitrine) in a buffer (PBS, pH 7.4) at room temperature. Complementary experiments were carried out on free CFP, YFP, and the cleaved FRET probe as controls. The wavelength-dependent fluorescence lifetime measurements of the donor and acceptor in the FRET probe, using the time-correlated single-photon counting technique, indicate an energy transfer efficiency of 6.8 ± 0.9% in PBS, with distinct excited-state dynamics from the recombinant CFP and YFP. The estimated mCerulean-mCitrine distance in this FRET probe is 7.7 ± 0.2 nm. The energy transfer efficiency increases (11.5 ± 0.9%) as the concentration of Ficoll-70 increases over the range of 0-300 g/L with an estimated mCerulean-mCitrine distance of 6.1 ± 0.2 nm. Complementary time-resolved anisotropy measurements suggest that the rotational diffusion of hetero-FRET in PBS is sensitive to the energy transfer from the donor to the acceptor. The results also suggest that the linker, -(GSG) 6 A(EAAAK) 6 A(GSG) 6 A(EAAAK) 6 A(GSG) 6 -, is rather flexible, and the observed rotational dynamics is likely to be due to a segmental mobility of the FRET pairs rather than an overall tumbling motion of a rigid probe. Comparative studies on a new construct of a FRET probe with a shorter, more flexible linker, mCerulean-(GSG) 18 -mCitrine, reveal enhanced energy transfer efficiency. On the millisecond time scale, fluorescence fluctuation analyses of the acceptor (excited at 488 nm) provide a means to examine the translational diffusion coefficient of the FRET probe. The results also suggest that the linker is flexible in this FRET probe, and the observed diffusion coefficient is faster than predicted as compared to the cleaved FRET probe. Our results serve as a point of reference for this FRET probe in a buffer toward its full potential as a sensor for macromolecular crowding in living cells and tissues.

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 Förster 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 FRET-increases 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., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Macromolecular crowding affects the mobility of biomolecules, protein folding and stability, and the association of macromolecules with each other. Local differences in crowding that arise as a result of subcellular components and supramolecular assemblies contribute to the structural organization of the cytoplasm. In this Opinion article we discuss how macromolecular crowding affects the physicochemistry of the cytoplasm and how this, in turn, affects microbial physiology. We propose that cells maintain the overall concentration of macromolecules within a narrow range and discuss possible mechanisms for achieving crowding homeostasis. In addition, we propose that the term 'homeocrowding' is used to describe the process by which cells maintain relatively constant levels of macromolecules.

Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric α-hemolysin pore (αHL) has been engineered to form a nanoreactor to study covalent chemistry at the single-molecule level. Previous work has been confined largely to the chemistry of cysteine side chains or, in one instance, to an irreversible reaction of an unnatural amino acid side chain bearing a terminal alkyne. Here, we present four different αHL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic αHL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side chain at the single-molecule level.

The cytosol of Escherichia coli is an extremely crowded environment, containing high concentrations of biopolymers which occupy 20-30% of the available volume. Such conditions are expected to yield depletion forces, which strongly promote macromolecular complexation. However, crowded macromolecule solutions, like the cytosol, are very prone to nonspecific associative interactions that can potentially counteract depletion. It remains unclear how the cytosol balances these opposing interactions. We used a FRET-based probe to systematically study depletion in vitro in different crowded environments, including a cytosolic mimic, E. coli lysate. We also studied bundle formation of FtsZ protofilaments under identical crowded conditions as a probe for depletion interactions at much larger overlap volumes of the probe molecule. The FRET probe showed a more compact conformation in synthetic crowding agents, suggesting strong depletion interactions. However, depletion was completely negated in cell lysate and other protein crowding agents, where the FRET probe even occupied slightly more volume. In contrast, bundle formation of FtsZ protofilaments proceeded as readily in E. coli lysate and other protein solutions as in synthetic crowding agents. Our experimental results and model suggest that, in crowded biopolymer solutions, associative interactions counterbalance depletion forces for small macromolecules. Furthermore, the net effects of macromolecular crowding will be dependent on both the size of the macromolecule and its associative interactions with the crowded background.

Macromolecular crowding in cells influences processes such as folding, association and diffusion of proteins and polynucleic acids. Direct spatiotemporal readout of crowding would be a powerful approach for unraveling the structure of the cytoplasm and determining the impact of excluded volume on protein function in living cells. Here, we introduce a genetically encodable fluorescence resonance energy transfer (FRET) sensor for quantifying macromolecular crowding and discuss our application of the sensor in bacterial and mammalian cells.

Interactions of the azachalcone derived substrate Aza with copper(II) complexes in the presence and absence of st-DNA were studied in detail by UV/Vis absorption, EPR and Raman and (UV and vis) resonance Raman spectroscopies. The binding of Aza to the Lewis acidic copper(II) complexes, which results in activation of the substrate, was established spectroscopically. It was shown that the binding of Aza differs between Cu(II)dmbpy and Cu(II)terpy, consistent with the observed differences in catalytic asymmetric Diels-Alder reactions with regard to both the rate and enantiomeric preference. Finally, it was shown that DNA has a major beneficial effect on the binding of Aza to the copper(II) complex due to the fact that both bind to the DNA. The result is a high effective molarity of both the copper complexes and the Aza substrate, which leads to a significant increase in binding of Aza to the copper(II) complex. This effect is a key reason for the observed rate acceleration in the catalyzed reactions brought about by the presence of DNA.

The interaction between salmon testes DNA (st-DNA) and a series of Cu(II) polypyridyl complexes, i.e. [Cu(dmbpy)(NO3)2] (1) (dmbpy = 4,4'-dimethyl-2,2'-bipyridine), [Cu(bpy)(NO3)2] (2) (bpy = 2,2'-bipyridine), [Cu(phen)(NO3)2] (3) (phen = phenanthroline), [Cu(terpy)(NO3)2]·H2O (4) (terpy = 2,2':6',2″-terpyridine), [Cu(dpq)(NO3)2] (5) (dpq = dipyrido-[3,2-d:2',3'-f]-quinoxaline) and [Cu(dppz)(NO3)2] (6) (dppz = dipyrido[3,2-a:2',3'-c]phenazine) was studied by UV/Vis absorption, Circular Dichroism, Linear Dichroism, EPR, Raman and (UV and vis) resonance Raman spectroscopies and viscometry. These complexes catalyse enantioselective C-C bond forming reactions in water with DNA as the source of chirality. Complex 1 crystallizes as an inorganic polymer with nitrate ligands bridging the copper ions, which adopt essentially a distorted square pyramidal structure with a fifth bridging nitrate ligand at the axial position. Raman spectroscopy indicates that in solution the nitrate ligands in 1, 2, 3 and 4 are displaced by solvent (H2O). For complex 1, multiple supramolecular species are observed in the presence of st-DNA in contrast to the other complexes, which appear to interact relatively uniformly as a single species predominantly, when st-DNA is present. Overall the data suggest that complexes 1 and 2 engage primarily through groove binding with st-DNA while 5 and 6 undergo intercalation. For complexes 3 and 4 the data indicates that both groove binding and intercalation takes place, albeit primarily intercalation. Although it is tempting to conclude that the groove binders give highest ee and rate acceleration, it is proposed that the flexibility and dynamics in binding of Cu(II) complexes to DNA are key parameters that determine the outcome of the reaction. These findings provide insight into the complex supramolecular structure of these DNA-based catalysts.

The detection of several different neurotransmitters with the same sensor in real-time would be a powerful asset to the field of neurochemistry. We have developed a detector for a broad range of neurotransmitters including amino acids, catecholamines, and nucleotides, which relies on the reversible binding of the analytes to a copper(II) complex within an engineered protein nanopore.

DNA-based catalysis can be used to control the enantioselectivity of copper-catalysed Diels-Alder and Friedel-Crafts reactions to produce either enantiomer of the product by changing the denticity of the ligand coordinated to the Cu(II) ion, even though the DNA adopts a right handed helical conformation only., (This journal is © The Royal Society of Chemistry 2012)

The enantioselective addition of water to olefins in an aqueous environment is a common transformation in biological systems, but was beyond the ability of synthetic chemists. Here, we present the first examples of a non-enzymatic catalytic enantioselective hydration of enones, for which we used a catalyst that comprises a copper complex, based on an achiral ligand, non-covalently bound to (deoxy)ribonucleic acid, which is the only source of chirality present under the reaction conditions. The chiral β-hydroxy ketone product was obtained in up to 82% enantiomeric excess. Deuterium-labelling studies demonstrated that the reaction is diastereospecific, with only the syn hydration product formed. So far, this diastereospecific and enantioselective reaction had no equivalent in conventional homogeneous catalysis.

A kinetic study of DNA-based catalytic enantioselective Friedel-Crafts alkylation and Michael addition reactions showed that DNA affects the rate of these reactions significantly. Whereas in the presence of DNA, a large acceleration was found for the Friedel-Crafts alkylation and a modest acceleration in the Michael addition of dimethyl malonate, a deceleration was observed when using nitromethane as nucleophile. Also, the enantioselectivities proved to be dependent on the DNA sequence. In comparison with the previously reported Diels-Alder reaction, the results presented here suggest that DNA plays a similar role in both cycloaddition and conjugate addition reactions.

The unique chiral structure of DNA has been a source of inspiration for the development of a new class of bio-inspired catalysts. The novel concept of DNA-based asymmetric catalysis, which was introduced only five years ago, has been applied successfully in a variety of catalytic enantioselective reactions. In this tutorial review, the ideas behind this novel concept will be introduced, an overview of the catalytic chemistry available to date will be given and the role of DNA in catalysis will be discussed. Finally, an overview of new developments of potential interest for DNA-based asymmetric catalysis will be provided.

The recently developed concept of DNA-based asymmetric catalysis involves the transfer of chirality from the DNA double helix in reactions using a noncovalently bound catalyst. To date, two generations of DNA-based catalysts have been reported that differ in the design of the ligand for the metal. Herein we present a study of the first generation of DNA-based catalysts, which contain ligands comprising a metal-binding domain linked through a spacer to a 9-aminoacridine moiety. Particular emphasis has been placed on determining the effect of DNA on the structure of the Cu(II) complex and the catalyzed Diels-Alder reaction. The most important findings are that the role of DNA is limited to being a chiral scaffold; no rate acceleration was observed in the presence of DNA. Furthermore, the optimal DNA sequence for obtaining high enantioselectivities proved to contain alternating GC nucleotides. Finally, DNA has been shown to interact with the Cu(II) complex to give a chiral structure. Comparison with the second generation of DNA-based catalysts, which bear bipyridine-type ligands, revealed marked differences, which are believed to be related to the DNA microenvironment in which the catalyst resides and where the reaction takes place.