Qiong Wu

19F NMR Spectroscopy as a Probe of Cytoplasmic Viscosity and Weak Protein Interactions in Living Cells

Protein mobility in living cells is vital for cell function. Both cytosolic viscosity and weak protein-protein interactions affect mobility, but examining viscosity and weak interaction effects is challenging. Herein, we demonstrate the use of (19) F NMR spectroscopy to measure cytoplasmic viscosity and to characterize nonspecific protein-protein interactions in living Escherichia coli cells. The origins of resonance broadening in Escherichia coli cells were also investigated. We found that sample inhomogeneity has a negligible effect on resonance broadening, the cytoplasmic viscosity is only about 2-3 times that of water, and ubiquitous transient weak protein-protein interactions in the cytosol play a significant role in governing the detection of proteins by using in-cell NMR spectroscopy.

Strategies for Protein NMR in Escherichia coli

In-cell NMR spectroscopy provides insight into protein conformation, dynamics, and function at atomic resolution in living cells. Systematic evaluation of isotopic-labeling strategies is necessary to observe the target protein in the sea of other molecules in the cell. Here, we investigate the detectability, sensitivity, and resolution of in-cell NMR spectra of the globular proteins GB1, ubiquitin, calmodulin, and bcl-xl-cutloop, resulting from uniform (15)N enrichment (with and without deuteration), selective (15)N-Leu enrichment, (13)C-methyl enrichment of isoleucine, leucine, valine, and alanine, fractional (13)C enrichment, and (19)F labeling. Most of the target proteins can be observed by (19)F labeling and (13)C enrichment with direct detection because selectively labeling suppresses background signals and because deuteration improves in-cell spectra. Our results demonstrate that the detectability of proteins is determined by weak interactions with intercellular components and that choosing appropriate labeling strategies is critical for the success of in-cell protein NMR studies.

Magnetic Resonance Spectroscopy as a Tool for Assessing Macromolecular Structure and Function in Living Cells

Investigating the structure, modification, interaction, and function of biomolecules in their native cellular environment leads to physiologically relevant knowledge about their mechanisms, which will benefit drug discovery and design. In recent years, nuclear and electron magnetic resonance (NMR) spectroscopy has emerged as a useful tool for elucidating the structure and function of biomacromolecules, including proteins, nucleic acids, and carbohydrates in living cells at atomic resolution. In this review, we summarize the progress and future of in-cell NMR as it is applied to proteins, nucleic acids, and carbohydrates.

Confinement Alters the Structure and Function of Calmodulin

Many cellular reactions involving proteins, including their biosynthesis, misfolding, and transport, occur in confined compartments. Despite its importance, a structural basis of understanding of how confined environments alter protein function is still lacking. Herein, we explore structure-function correlations of calmodulin (CaM), a multidomain protein involved in many calcium-mediated signaling pathways, in reverse micelles. Confinement dramatically alters CaM structure and function. The protein forms an extended structure in bulk water, but becomes compacted in reverse micelles. In addition, confinement changes the function of CaM. Specifically, the protein binds the MLCK, AcN19, and somatostatin peptides in dilute buffer, but binds only the MLCK and AcN19 peptides in reverse micelles. In summary, we determined a new CaM structure in reverse micelles and demonstrate that confinement can modulate both protein structure and function.

A dual fluorogenic and 19F NMR probe for the detection of esterase activity

In this study, a dual-channel probe of FlE based on flavonoid derivatives is reported, which can yield “turn-on” signals of both fluorometry and 19F nuclear magnetic resonance in response to the presence of esterase. Upon the addition of esterase, FlE could convert into Fl, possessing the ESIPT effect. As a result, the fluorescence intensity was significantly enhanced and peaked at 510 nm, accompanying a change in the fluorescence of solution from nonluminous to strong green. Moreover, as determined by 19F NMR, the signal could apparently shift from δF −111.57 to −111.69 ppm. Due to the combination of these two detection approaches having good sensitivity, high selectivity, and real-time detection, FlE has been successfully applied to confocal fluorescence imaging for the detection of esterase in cells, which consistent with the 19F NMR test results in some sense.

Quantification of size effect on protein rotational mobility in cells by 19F NMR spectroscopy

AbstractProtein diffusion in living cells might differ significantly from that measured in vitro. Little is known about the effect of globular protein size on rotational diffusion in cells because each protein has distinct surface properties, which result in different interactions with cellular components. To overcome this problem, the B1 domain of protein G (GB1) and several concatemers of the protein were labeled with 5-fluorotryptophan and studied by 19F NMR in Escherichia coli cells, Xenopus laevis oocytes, and in aqueous solutions crowded with glycerol, or Ficoll70™ and lysozyme. Relaxation data show that the size dependence of protein rotation in cells is due to weak interactions of the target protein with cellular components, but the effect of these interactions decreases as protein size increases. The results provide valuable information for interpreting protein diffusion data acquired in living cells.n Graphical abstractSize matters. The protein rotational mobility in living cells was assessed by 19F NMR. The size dependence effect may arise from weak interactions between protein and cytoplasmic components.

A pH-gated conformational switch regulates the phosphatase activity of bifunctional HisKA-family histidine kinases

Histidine kinases are key regulators in the bacterial two-component systems that mediate the cellular response to environmental changes. The vast majority of the sensor histidine kinases belong to the bifunctional HisKA family, displaying both kinase and phosphatase activities toward their substrates. The molecular mechanisms regulating the opposing activities of these enzymes are not well understood. Through a combined NMR and crystallographic study on the histidine kinase HK853 and its response regulator RR468 from Thermotoga maritima, here we report a pH-mediated conformational switch of HK853 that shuts off its phosphatase activity under acidic conditions. Such a pH-sensing mechanism is further demonstrated in the EnvZ-OmpR two-component system from Salmonella enterica in vitro and in vivo, which directly contributes to the bacterial infectivity. Our finding reveals a broadly conserved mechanism that regulates the phosphatase activity of the largest family of bifunctional histidine kinases in response to the change of environmental pH.Bacteria adapt to changing environmental conditions through signal transduction mediated by the two-component system (TCS). Here, the authors combine X-ray crystallography and NMR studies to characterize a pH-gated conformational switch that regulates the phosphatase activity of TCS bifunctional histidine kinases.

A 19F NMR probe for the detection of β-galactosidase: simple structure with low molecular weight of 274.2, “turn-on” signal without the background, and good performance applicable in cancer cell line

Based on the efficient cleavage reaction of the C-O ether bond triggered by β-gal selectively, FB-βGal, with good water-solubility, low toxicity, high specificity, excellent water-solubility and high biocompatibility, was prepared, which could report the presence of trace β-gal quickly and conveniently by a significant change in the 19F NMR spectra without any background noise. The successful application of FB-βGal for the detection of β-gal in living Escherichia coli, HeLa cells and OVCAR-3 cells quantitatively makes it a promising candidate for practical application in related fields.

Crowding and confinement can oppositely affect protein stability

Proteins encounter crowded and confined macromolecular milieus in living cells. Simple theory predicts that both environments entropically stabilize proteins if only hard-core repulsive interactions are considered. Recent studies show that chemical interactions between the surroundings and the test protein also play key roles such that the overall effect of crowding or confinement is a balance of hard-core repulsions and chemical interactions. There are, however, few quantitative studies. Here, we quantify the effects of crowding and confinement on the equilibrium unfolding thermodynamics of a model globular protein, KH1. The results do not agree with predictions from simple theory. KH1 is stabilized by synthetic-polymer crowding agents but destabilized by confinement in reverse micelles. KH1 is more entropically stabilized and enthalpically destabilized in concentrated solutions of the monomers than it is in solutions of the corresponding polymers. When KH1 is confined in reverse micelles, the temperature of maximum stability decreases, the melting temperature decreases, and the protein is entropically destabilized and enthalpically stabilized. Our results show the importance of chemical interactions to protein folding thermodynamics and imply that cells utilize chemical interactions to tune protein stability.

The Effects of Macromolecular Crowding on Calmodulin Structure and Function

Macromolecular crowding and confinement are two factors that potentially affect protein structure and function in a complex cellular environment. The confinement effect on the structure and function of holoCaM [Ca2+ -loaded calmodulin (CaM)], a two-domain protein involved in many calcium-mediated signaling pathways, has been investigated previously. However, little is known about how macromolecular crowding affects holoCaM structure and function. Here, the structure-function correlations of holoCaM are investigated in macromolecular crowded environments. It was found that macromolecular crowding impacts its structure and function mildly. The major conformational states are still extended conformation with inter-domain separation in crowded environment as well as those in dilute solution, but the population of transiently compact conformation increases compared to dilute solution. Furthermore, macromolecular crowding facilitates the binding of CaM with AcN19 peptide (CaM-bind domain of α-syn). This study provides a direct comparison for macromolecular crowding and confinement effects on protein structure and function, which helps to understand chemistry regulation in living cells.