An antibiotic from an uncultured bacterium binds to an immutable target.

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

The Structure of the Necrosome RIPK1-RIPK3 Core, a Human Hetero-Amyloid Signaling Complex.

The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis, serving in human immune defense, cancer, and neurodegenerative diseases. RIPK1 and RIPK3 associate through their RIP homotypic interaction motifs with consensus sequences IQIG (RIPK1) and VQVG (RIPK3). Using solid-state nuclear magnetic resonance, we determined the high-resolution structure of the RIPK1-RIPK3 core. RIPK1 and RIPK3 alternately stack (RIPK1, RIPK3, RIPK1, RIPK3, etc.) to form heterotypic ß sheets. Two such ß sheets bind together along a compact hydrophobic interface featuring an unusual ladder of alternating Ser (from RIPK1) and Cys (from RIPK3). The crystal structure of a four-residue RIPK3 consensus sequence is consistent with the architecture determined by NMR. The RIPK1-RIPK3 core is the first detailed structure of a hetero-amyloid and provides a potential explanation for the specificity of hetero- over homo-amyloid formation and a structural basis for understanding the mechanisms of signal transduction.

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR.

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase.

Molecular basis for curvature formation in SepF polymerization.

The self-assembly of proteins into curved structures plays an important role in many cellular processes. One good example of this phenomenon is observed in the septum-forming protein (SepF), which forms polymerized structures with uniform curvatures. SepF is essential for regulating the thickness of the septum during bacteria cell division. In Bacillus subtilis, SepF polymerization involves two distinct interfaces, the ß-ß and α-α interfaces, which define the assembly unit and contact interfaces, respectively. However, the mechanism of curvature formation in this step is not yet fully understood. In this study, we employed solid-state NMR (SSNMR) to compare the structures of cyclic wild-type SepF assemblies with linear assemblies resulting from a mutation of G137 on the ß-ß interface. Our results demonstrate that while the sequence differences arise from the internal assembly unit, the dramatic changes in the shape of the assemblies depend on the α-α interface between the units. We further provide atomic-level insights into how the angular variation of the α2 helix on the α-α interface affects the curvature of the assemblies, using a combination of SSNMR, cryo-electron microscopy, and simulation methods. Our findings shed light on the shape control of protein assemblies and emphasize the importance of interhelical contacts in retaining curvature.

Unraveling the molecular mechanism for enhanced gas adsorption in mixed-metal MOFs via solid-state NMR spectroscopy.

The incorporation of multiple metal ions in metal-organic frameworks (MOFs) through one-pot synthesis can induce unique properties originating from specific atomic-scale spatial apportionment, but the extraction of this crucial information poses challenges. Herein, nondestructive solid-state NMR spectroscopy was used to discern the atomic-scale metal apportionment in a series of bulk Mg1-xCox-MOF-74 samples via identification and quantification of eight distinct arrangements of Mg/Co ions labeled with a 13C-carboxylate, relative to Co content. Due to the structural characteristics of metal-oxygen chains, the number of metal permutations is infinite for Mg1-xCox-MOF-74, making the resolution of atomic-scale metal apportionment particularly challenging. The results were then employed in density functional theory calculations to unravel the molecular mechanism underlying the macroscopic adsorption properties of several industrially significant gases. It is found that the incorporation of weak adsorption sites (Mg2+ for CO and Co2+ for CO2 adsorption) into the MOF structure counterintuitively boosts the gas adsorption energy on strong sites (Co2+ for CO and Mg2+ for CO2 adsorption). Such effect is significant even for Co2+ remote from Mg2+ in the metal-oxygen chain, resulting in a greater enhancement of CO adsorption across a broad composition range, while the enhancement of CO2 adsorption is restricted to Mg2+ with adjacent Co2+. Dynamic breakthrough measurements unambiguously verified the trend in gas adsorption as a function of metal composition. This research thus illuminates the interplay between atomic-scale structures and macroscopic gas adsorption properties in mixed-metal MOFs and derived materials, paving the way for developing superior functional materials.

Structures of AT8 and PHF1 phosphomimetic tau: Insights into the posttranslational modification code of tau aggregation.

The microtubule-associated protein tau aggregates into amyloid fibrils in Alzheimer's disease and other neurodegenerative diseases. In these tauopathies, tau is hyperphosphorylated, suggesting that this posttranslational modification (PTM) may induce tau aggregation. Tau is also phosphorylated in normal developing brains. To investigate how tau phosphorylation induces amyloid fibrils, here we report the atomic structures of two phosphomimetic full-length tau fibrils assembled without anionic cofactors. We mutated key Ser and Thr residues to Glu in two regions of the protein. One construct contains three Glu mutations at the epitope of the anti-phospho-tau antibody AT8 (AT8-3E tau), whereas the other construct contains four Glu mutations at the epitope of the antibody PHF1 (PHF1-4E tau). Solid-state NMR data show that both phosphomimetic tau mutants form homogeneous fibrils with a single set of chemical shifts. The AT8-3E tau rigid core extends from the R3 repeat to the C terminus, whereas the PHF1-4E tau rigid core spans R2, R3, and R4 repeats. Cryoelectron microscopy data show that AT8-3E tau forms a triangular multi-layered core, whereas PHF1-4E tau forms a triple-stranded core. Interestingly, a construct combining all seven Glu mutations exhibits the same conformation as PHF1-4E tau. Scalar-coupled NMR data additionally reveal the dynamics and shape of the fuzzy coat surrounding the rigid cores. These results demonstrate that specific PTMs induce structurally specific tau aggregates, and the phosphorylation code of tau contains redundancy.

Contribution of protein conformational heterogeneity to NMR lineshapes at cryogenic temperatures.

While low-temperature Nuclear Magnetic Resonance (NMR) holds great promise for the analysis of unstable samples and for sensitizing NMR detection, spectral broadening in frozen protein samples is a common experimental challenge. One hypothesis explaining the additional linewidth is that a variety of conformations are in rapid equilibrium at room temperature and become frozen, creating an inhomogeneous distribution at cryogenic temperatures. Here, we investigate conformational heterogeneity by measuring the backbone torsion angle (Ψ) in Escherichia coli Dihydrofolate Reductase (DHFR) at 105 K. Motivated by the particularly broad N chemical shift distribution in this and other examples, we modified an established NCCN Ψ experiment to correlate the chemical shift of Ni+1 to Ψi. With selective 15N and 13C enrichment of Ile, only the unique I60-I61 pair was expected to be detected in 13C'-15N correlation spectrum. For this unique amide, we detected three different conformation basins based on dispersed chemical shifts. Backbone torsion angles Ψ were determined for each basin: 114 ± 7° for the major peak and 150 ± 8° and 164 ± 16° for the minor peaks as contrasted with 118° for the X-ray crystal structure (and 118° to 130° for various previously reported structures). These studies support the hypothesis that inhomogeneous distributions of protein backbone torsion angles contribute to the lineshape broadening in low-temperature NMR spectra.

Correlating local structure and migration dynamics in Na/Li dual ion conductor Na5YSi4O12.

Na5YSi4O12 (NYSO) is demonstrated as a promising electrolyte with high ionic conductivity and low activation energy for practical use in solid Na-ion batteries. Solid-state NMR was employed to identify the six types of coordination of Na+ ions and migration pathway, which is vital to master working mechanism and enhance performance. The assignment of each sodium site is clearly determined from high-quality 23Na NMR spectra by the aid of Density Functional Theory calculation. Well-resolved 23Na exchangespectroscopy and electrochemical tracer exchange spectra provide the first experimental evidence to show the existence of ionic exchange between sodium at Na5 and Na6 sites, revealing that Na transport route is possibly along three-dimensional chain of open channel-Na4-open channel. Variable-temperature NMR relaxometry is developed to evaluate Na jump rates and self-diffusion coefficient to probe the sodium-ion dynamics in NYSO. Furthermore, NYSO works well as a dual ion conductor in Na and Li metal batteries with Na3V2(PO4)3 and LiFePO4 as cathodes, respectively.

Atomic resolution structure of full-length human insulin fibrils.

Patients with type 1 diabetes mellitus who are dependent on an external supply of insulin develop insulin-derived amyloidosis at the sites of insulin injection. A major component of these plaques is identified as full-length insulin consisting of the two chains A and B. While there have been several reports that characterize insulin misfolding and the biophysical properties of the fibrils, atomic-level information on the insulin fibril architecture remains elusive. We present here an atomic resolution structure of a monomorphic insulin amyloid fibril that has been determined using magic angle spinning solid-state NMR spectroscopy. The structure of the insulin monomer yields a U-shaped fold in which the two chains A and B are arranged in parallel to each other and are oriented perpendicular to the fibril axis. Each chain contains two ß-strands. We identify two hydrophobic clusters that together with the three preserved disulfide bridges define the amyloid core structure. The surface of the monomeric amyloid unit cell is hydrophobic implicating a potential dimerization and oligomerization interface for the assembly of several protofilaments in the mature fibril. The structure provides a starting point for the development of drugs that bind to the fibril surface and disrupt secondary nucleation as well as for other therapeutic approaches to attenuate insulin aggregation.

Structure of the nonhelical filament of the Alzheimer's disease tau core.

The microtubule-associated protein tau aggregates into neurofibrillary tangles in Alzheimer's disease (AD). The main type of aggregates, the paired helical filaments (PHF), incorporate about 20% of the full-length protein into the rigid core. Recently, cryo-electron microscopy data showed that a protease-resistant fragment of tau (residues 297-391) self-assembles in vitro in the presence of divalent cations to form twisted filaments whose molecular structure resembles that of AD PHF tau [S. Lövestam et al., Elife 11, e76494 (2022)]. To investigate whether this tau construct is uniquely predisposed to this morphology and structure, we fibrillized tau (297-391) under the reported conditions and determined its structure using solid-state NMR spectroscopy. Unexpectedly, the protein assembled predominantly into nontwisting ribbons whose rigid core spans residues 305-357. This rigid core forms a ß-arch that turns at residues 322CGS324. Two protofilaments stack together via a long interface that stretches from G323 to I354. Together, these two protofilaments form a four-layered ß-sheet core whose sidechains are stabilized by numerous polar and hydrophobic interactions. This structure gives insight into the fibril morphologies and molecular conformations that can be adopted by this protease-resistant core of AD tau under different pH and ionic conditions.

Solid-state NMR molecular snapshots of Aspergillus fumigatus cell wall architecture during a conidial morphotype transition.

While establishing an invasive infection, the dormant conidia of Aspergillus fumigatus transit through swollen and germinating stages, to form hyphae. During this morphotype transition, the conidial cell wall undergoes dynamic remodeling, which poses challenges to the host immune system and antifungal drugs. However, such cell wall reorganization during conidial germination has not been studied so far. Here, we explored the molecular rearrangement of Aspergillus fumigatus cell wall polysaccharides during different stages of germination. We took advantage of magic-angle spinning NMR to investigate the cell wall polysaccharides, without employing any destructive method for sample preparation. The breaking of dormancy was associated with a significant change in the molar ratio between the major polysaccharides ß-1,3-glucan and α-1,3-glucan, while chitin remained equally abundant. The use of various polarization transfers allowed the detection of rigid and mobile polysaccharides; the appearance of mobile galactosaminogalactan was a molecular hallmark of germinating conidia. We also report for the first time highly abundant triglyceride lipids in the mobile matrix of conidial cell walls. Water to polysaccharides polarization transfers revealed an increased surface exposure of glucans during germination, while chitin remained embedded deeper in the cell wall, suggesting a molecular compensation mechanism to keep the cell wall rigidity. We complement the NMR analysis with confocal and atomic force microscopies to explore the role of melanin and RodA hydrophobin on the dormant conidial surface. Exemplified here using Aspergillus fumigatus as a model, our approach provides a powerful tool to decipher the molecular remodeling of fungal cell walls during their morphotype switching.

Structures of brain-derived 42-residue amyloid-ß fibril polymorphs with unusual molecular conformations and intermolecular interactions.

Fibrils formed by the 42-residue amyloid-ß peptide (Aß42), a main component of amyloid deposits in Alzheimer's disease (AD), are known to be polymorphic, i.e., to contain multiple possible molecular structures. Previous studies of Aß42 fibrils, including fibrils prepared entirely in vitro or extracted from brain tissue and using solid-state NMR (ssNMR) or cryogenic electron microscopy (cryo-EM) methods, have found polymorphs with differences in amino acid sidechain orientations, lengths of structurally ordered segments, and contacts between cross-ß subunit pairs within a single filament. Despite these differences, Aß42 molecules adopt a common S-shaped conformation in all previously described high-resolution Aß42 fibril structures. Here we report two cryo-EM-based structures of Aß42 fibrils that are qualitatively different, in samples derived from AD brain tissue by seeded growth. In type A fibrils, residues 12 to 42 adopt a ν-shaped conformation, with both intra-subunit and intersubunit hydrophobic contacts to form a compact core. In type B fibrils, residues 2 to 42 adopt an υ-shaped conformation, with only intersubunit contacts and internal pores. Type A and type B fibrils have opposite helical handedness. Cryo-EM density maps and molecular dynamics simulations indicate intersubunit K16-A42 salt bridges in type B fibrils and partially occupied K28-A42 salt bridges in type A fibrils. The coexistence of two predominant polymorphs, with differences in N-terminal dynamics, is supported by ssNMR data, as is faithful propagation of structures from first-generation to second-generation brain-seeded Aß42 fibril samples. These results demonstrate that Aß42 fibrils can exhibit a greater range of structural variations than seen in previous studies.

A prototype protein nanocage minimized from carboxysomes with gated oxygen permeability.

Protein nanocages (PNCs) in cells and viruses have inspired the development of self-assembling protein nanomaterials for various purposes. Despite the successful creation of artificial PNCs, the de novo design of PNCs with defined permeability remains challenging. Here, we report a prototype oxygen-impermeable PNC (OIPNC) assembled from the vertex protein of the ß-carboxysome shell, CcmL, with quantum dots as the template via interfacial engineering. The structure of the cage was solved at the atomic scale by combined solid-state NMR spectroscopy and cryoelectron microscopy, showing icosahedral assembly of CcmL pentamers with highly conserved interpentamer interfaces. Moreover, a gating mechanism was established by reversibly blocking the pores of the cage with molecular patches. Thus, the oxygen permeability, which was probed by an oxygen sensor inside the cage, can be completely controlled. The CcmL OIPNC represents a PNC platform for oxygen-sensitive or oxygen-responsive storage, catalysis, delivery, sensing, etc.

Probing Watson-Crick and Hoogsteen base pairing in duplex DNA using dynamic nuclear polarization solid-state NMR spectroscopy.

The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.

Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR.

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the ß-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the ß-subunit from solution.

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Solid-state NMR MAS CryoProbe enables structural studies of human blood protein vitronectin bound to hydroxyapatite.

The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments. Vn is a major blood protein that regulates many different physiological and pathological processes. The high sensitivity of the CryoProbe enabled us to acquire three-dimensional solid-state NMR spectra for sequential assignment and characterization of site-specific water-protein interactions that provide initial insights into the organization of the Vn-HAP complex. Vn associates with HAP in various pathological settings, including macular degeneration eyes and Alzheimer's disease brains. The ability to probe these assemblies at atomic detail paves the way for understanding their formation.

An electrostatic cluster guides Aß40 fibril formation in sporadic and Dutch-type cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is associated with the accumulation of fibrillar Aß peptides upon and within the cerebral vasculature, which leads to loss of vascular integrity and contributes to disease progression in Alzheimer's disease (AD). We investigate the structure of human-derived Aß40 fibrils obtained from patients diagnosed with sporadic or familial Dutch-type (E22Q) CAA. Using cryo-EM, two primary structures are identified containing elements that have not been observed in in vitro Aß40 fibril structures. One population has an ordered N-terminal fold comprised of two ß-strands stabilized by electrostatic interactions involving D1, E22, D23 and K28. This charged cluster is disrupted in the second population, which exhibits a disordered N-terminus and is favored in fibrils derived from the familial Dutch-type CAA patient. These results illustrate differences between human-derived CAA and AD fibrils, and how familial CAA mutations can guide fibril formation.

Modulation of aggregation and structural polymorphisms of ß-amyloid fibrils in cellular environments by pyroglutamate-3 variant cross-seeding.

Amyloidogenic deposition of ß-amyloid (Aß) peptides in human brain involves not only the wild-type Aß (wt-Aß) sequences, but also posttranslationally modified Aß (PTM-Aß) variants. Recent studies hypothesizes that the PTM-Aß variants may trigger the deposition of wt-Aß, which underlies the pathology of Sporadic Alzheimer's disease. Among PTM-Aß variants, the pyroglutamate-3-Aß (pyroE3-Aß) has attracted much attention because of their significant abundances and broad distributions in senile plaques and dispersible and soluble oligomers. pyroE3-specific antibodies are being tested as potential anti-Aß drugs in clinical trials. However, evidence that support the triggering effect of pyroE3-Aß on wt-Aß in cells remain lacking, which diminishes its pathological relevance. We show here that cross-seeding with pyroE3-Aß40 leads to accelerated extracellular and intracellular aggregation of wt-Aß40 in different neuronal cells. Cytotoxicity levels are elevated through the cross-seeded aggregation, comparing with the self-seeded aggregation of wt-Aß40 or the static presence of pyroE3-Aß40 seeds. For the extracellular deposition in mouse neuroblastoma Neuro2a (N2a) cells, the cytotoxicity elevation correlates positively with the seeding efficiency. Besides aggregation rates, cross-seeding with pyroE3-Aß40 also modulates the molecular level structural polymorphisms of the resultant wt-Aß40 fibrils. Using solid-state nuclear magnetic resonance (ssNMR) spectroscopy, we identified key structural differences between the parent pyroE3/ΔE3 and wt-Aß40 fibrils within their fibrillar cores. Structural propagation from seeds to daughter fibrils is demonstrated to be more pronounced in the extracellular seeding in N2a cells by comparing the ssNMR spectra from different seeded wt-Aß40 fibrils, but less significant in the intracellular seeding process in human neuroblastoma SH-SY5Y cells.

DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular milieu.

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition. We found that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular constituents.