Dimer System Research Articles

Effects of All-Atom Molecular Mechanics Force Fields on Amyloid Peptide Assembly: The Case of PHF6 Peptide of Tau Protein.

Molecular dynamics (MD) simulations play a vital role in revealing the mechanism of amyloid aggregation that is crucial to the therapeutic agent development for Alzheimer's Disease. However, the accuracy of MD simulation results strongly depends on the force field employed. In our previous benchmark for 17 all-atom force fields on modeling of amyloid aggregation using the Aβ16-22 dimer, we showed that AMBER14SB and CHARMM36m are suitable force fields for amyloid aggregation simulation, while GROMOS54a7 and OPLSAA are not good for the task. In this work, we continue assessing the applicability of atomistic force fields on amyloid aggregation using the VQIVYK (PHF6) peptide which is essential for tau-protein aggregation. Although, both Aβ16-22 and PHF6 peptides formed fibrils in vitro, the PHF6 fibrils are parallel β-sheets, while the Aβ16-22 fibrils are antiparallel β-sheets. We performed an all-atom large-scale MD simulation in explicit water on the PHF6 dimer and octa-peptides systems using five mainstream force fields, including AMBER99SB-disp, AMBER14SB, CHARMM36m, GROMOS54a7, and OPLSAA. The accumulated simulation time is 0.2 ms. Our result showed that the β-sheet structures of PHF6 peptides sampled by AMBER99SB-disp, AMBER14SB, GROMOS54a7, and OPLSAA are in favor of the antiparallel β-sheets, while the dominant type of β-sheet structures is parallel β-sheet by using CHARMM36m. Among the five force fields, CHARMM36m provides the strongest CH-π interaction that was observed in an NMR study. The comparison between our results and experimental observation indicates that CHARMM36m achieved the best performance on modeling the aggregation of PHF6 peptides. In summary, CHARMM36m is currently the most suitable force field for studying the aggregation of both amyloid-β and Tau through MD simulations.

Spin-Pure Stochastic-CASSCF via GUGA-FCIQMC Applied to Iron-Sulfur Clusters.

In this work, we demonstrate how to efficiently compute the one- and two-body reduced density matrices within the spin-adapted full configuration interaction quantum Monte Carlo (FCIQMC) method, which is based on the graphical unitary group approach (GUGA). This allows us to use GUGA-FCIQMC as a spin-pure configuration interaction (CI) eigensolver within the complete active space self-consistent field (CASSCF) procedure and hence to stochastically treat active spaces far larger than conventional CI solvers while variationally relaxing orbitals for specific spin-pure states. We apply the method to investigate the spin ladder in iron–sulfur dimer and tetramer model systems. We demonstrate the importance of the orbital relaxation by comparing the Heisenberg model magnetic coupling parameters from the CASSCF procedure to those from a CI-only (CASCI) procedure based on restricted open-shell Hartree–Fock orbitals. We show that the orbital relaxation differentially stabilizes the lower-spin states, thus enlarging the coupling parameters with respect to the values predicted by ignoring orbital relaxation effects. Moreover, we find that, while CASCI results are well fit by a simple bilinear Heisenberg Hamiltonian, the CASSCF eigenvalues exhibit deviations that necessitate the inclusion of biquadratic terms in the model Hamiltonian.

Abstract MP20: Optogenetic Control Of Pip2 To Assess Mechanisms Regulating The Epithelial Sodium Channel

The epithelial Na + channel (ENaC) plays a key role in Na + transport in epithelial linings to include the lung, colon and kidney. In the distal kidney tubules, ENaC regulates Na + reabsorption and blood volume. Thus, dysfunctions in signaling pathways regulating ENaC activity are linked to hypertension or hypotension. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is a target of the G protein coupled receptor P2Y2 pathway, and is necessary for the proper function of ENaC. This nonvoltage-gated trimeric channel is comprised of α, β, and γ subunits. We recently described two intracellular PIP 2 binding sites on the N termini of β-, and γ-ENaC, with moderate μM affinity. Here, we report the functional effects on ENaC containing a combination of mutations to those PIP 2 binding sites, by controlled depletion of PIP 2 . We used a CIBN/CRY2-5-ptase optogenetic dimerization system to deplete PIP 2 levels in HEK293 cells transiently expressing wild type (wt) ENaC or the mutant ENaC constructs. A fluorescent Na + indicator, was used to monitor ENaC activity by tracking the relative intracellular Na + levels. Upon optogenetic-controlled depletion of PIP 2 , Na + levels decreased in cells expressing wt ENaC. Mutations to the PIP 2 sites of ENaC were expected to have no change in Na + levels upon PIP 2 depletion due to the disruption of PIP 2 binding. As a control, mutations to non-PIP 2 binding sites were included, and were expected to have decreased Na + levels similar to wt ENaC. Interestingly, mutation of each independent PIP 2 site resulted in only a small decrease of intracellular Na + , compared to wt ENaC. However, mutations throughout the entire N-terminus of β-ENaC, including the PIP 2 binding site, resulted in a significant increase of Na + upon PIP 2 depletion. We performed patch clamp electrophysiology and found that the ENaC recordings corresponded to the Na + fluctuations. These data suggest that the residues surrounding the PIP 2 binding sites play a significant role in the affinity of PIP 2 for ENaC. The role of these other domains in PIP 2 binding is still under investigation.

Triplon current generation in solids

A triplon refers to a fictitious particle that carries angular momentum S=1 corresponding to the elementary excitation in a broad class of quantum dimerized spin systems. Such systems without magnetic order have long been studied as a testing ground for quantum properties of spins. Although triplons have been found to play a central role in thermal and magnetic properties in dimerized magnets with singlet correlation, a spin angular momentum flow carried by triplons, a triplon current, has not been detected yet. Here we report spin Seebeck effects induced by a triplon current: triplon spin Seebeck effect, using a spin-Peierls system CuGeO3. The result shows that the heating-driven triplon transport induces spin current whose sign is positive, opposite to the spin-wave cases in magnets. The triplon spin Seebeck effect persists far below the spin-Peierls transition temperature, being consistent with a theoretical calculation for triplon spin Seebeck effects.

Neutron diffraction of field-induced magnon condensation in the spin-dimerized antiferromagnet Sr3Cr2O8

In this work, we investigate the evolution and settling of magnon condensation in the spin-1/2 dimer system ${\mathrm{Sr}}_{3}{\mathrm{Cr}}_{2}{\mathrm{O}}_{8}$ using a combination of magnetostriction in pulsed fields and inelastic neutron scattering in a continuous magnetic field. The magnetic structure in the Bose-Einstein condensation phase was probed by neutron diffraction in pulsed magnetic fields up to 39 T. The magnetic structure in this phase was confirmed to be an $\mathit{XY}$-antiferromagnetic structure validated by irreducible representational analysis. The magnetic phase diagram as a function of an applied magnetic field for this system is presented. Furthermore, zero-field neutron diffraction results indicate that dimerization plays an important role in stabilizing the low-temperature crystal structure.

Handcuffing intrinsically disordered regions in Mlh1-Pms1 disrupts mismatch repair.

The DNA mismatch repair (MMR) factor Mlh1–Pms1 contains long intrinsically disordered regions (IDRs) whose exact functions remain elusive. We performed cross-linking mass spectrometry to identify interactions within Mlh1–Pms1 and used this information to insert FRB and FKBP dimerization domains into their IDRs. Baker's yeast strains bearing these constructs were grown with rapamycin to induce dimerization. A strain containing FRB and FKBP domains in the Mlh1 IDR displayed a complete defect in MMR when grown with rapamycin. but removing rapamycin restored MMR functions. Strains in which FRB was inserted into the IDR of one MLH subunit and FKBP into the other subunit were also MMR defective. The MLH complex containing FRB and FKBP domains in the Mlh1 IDR displayed a rapamycin-dependent defect in Mlh1–Pms1 endonuclease activity. In contrast, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization inappropriately activated Mlh1–Pms1 endonuclease activity. We conclude that dynamic and coordinated rearrangements of the MLH IDRs both positively and negatively regulate how the MLH complex acts in MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.

Rapid manipulation of mitochondrial morphology in a living cell with iCMM

SummaryEngineered synthetic biomolecular devices that integrate elaborate information processing and precisely regulate living cell behavior have potential in various applications. Although devices that directly regulate key biomolecules constituting inherent biological systems exist, no devices have been developed to control intracellular membrane architecture, contributing to the spatiotemporal functions of these biomolecules. This study developed a synthetic biomolecular device, termed inducible counter mitochondrial morphology (iCMM), to manipulate mitochondrial morphology, an emerging informative property for understanding physiopathological cellular behaviors, on a minute timescale by using a chemically inducible dimerization system. Using iCMM, we determined cellular changes by altering mitochondrial morphology in an unprecedented manner. This approach serves as a platform for developing more sophisticated synthetic biomolecular devices to regulate biological systems by extending manipulation targets from conventional biomolecules to mitochondria. Furthermore, iCMM might serve as a tool for uncovering the biological significance of mitochondrial morphology in various physiopathological cellular processes.

Mem-iLID, a fast and economic protein purification method.

Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein–protein interaction.

Production of Universal Anti-Bcma CAR-T Cells with Reduced Alloreactivity, but Potent Effector Function for the Treatment of Multiple Myeloma

Chimeric antigen receptor (CAR)-T therapy has recently shown great promise against multiple myeloma (MM) by targeting the B cell maturation antigen (BCMA). However, the production of autologous CAR-T cells on an individualized basis has significant challenges such as manufacture time, reproducibility, consistency and cost. In addition, viral-vector-produced CAR-T cells tend to be comprised of differentiated T cell subsets, which correlate with poor in vivo durability. Using the high-fidelity NextGEN™ (NG) CRISPR gene editing system in combination with the non-viral piggyBac™ (PB) DNA transposition technology, which produces CAR-T cells predominantly in a highly desirable stem cell memory T cell subset, we have developed P-BCMA-ALLO1, an off-the-shelf allogeneic CAR-T cell product that overcomes these limitations and can be given to any MM patient.The NG system was used to disrupt both TCR and MHCI expression in P-BCMA-ALLO1 to reduce possible alloreactivities. The DNA cleavage specificity is guaranteed by a truly dimeric system requiring the simultaneous binding of two obligate homodimers. High-throughput DNA sequencing analysis was performed on human T cells with gene disruption of TCRα, β2M or PD-1. Indel frequencies of amplified genomic on-target and off-target sites were evaluated from T cells that had been edited with either single or dual guide RNAs (gRNA)s. Herein, an average of 100,000 sequences were analyzed per site and non-edited T cells were included as a background reference. For these targets, the NG system demonstrated high-efficiency gene editing (>84% for TCRα, >64% for β2M, and >50% for PD-1) that was performed in resting T cells. No indel events were detected at any off-target site, proving the exquisite specificity of this system.P-BCMA-ALLO1 was incapable of mediating allo-responses such as graft-vs-host disease (GvHD) and graft rejection. Using a clinically-scalable purification method, P-BCMA-ALLO1 cells were enriched to be >99.5% TCR/MHCI double-negative and alloreactivities were evaluated by standard mixed lymphocyte reaction for either cell proliferation or IFNγ production in two separate assays: a 7-12 day CFSE-based cell proliferation assay and a 20-hour IFNγ-ELISPOT assay. Compared to non-edited CAR-T cells, P-BCMA-ALLO1 did not exhibit any detectable response to allogeneic PBMC stimulation, nor did it trigger any allogeneic T cell proliferative response above background level of cell proliferation or IFNγ production.Pre-clinical evaluation of P-BCMA-ALLO1 demonstrated optimal T cell phenotype and potent in vitro and in vivo efficacy. More than 95% of the P-BCMA-ALLO1 cells were CAR+ and greater than 50% of the product were CD45RA+CD62L+ stem cell memory T cells (TSCM), a highly desirable early memory T cell subset that exhibits the greatest capacity for self-renewal and the multipotent capacity to the entire spectrum of memory and effector T cell subsets. In addition, the CAR-T cells did not express the exhaustion markers PD-1, TIM-3 and LAG-3, nor did they exhibit any evidence of tonic signaling as IFNγ production in the absence of target antigen. In a short-term 24-hour in vitro co-culture assay with BCMA+ MM cell lines, P-BCMA-ALLO1 secreted high levels of IFNγ and mediated robust (70-90%) killing of cell targets. In addition, more than 80% of the cells proliferated in a longer 72-hour CFSE-based proliferation assay. P-BCMA-ALLO1 also showed strong potency in a NSG mouse model engrafted with MM.1S cells, an aggressive human MM-derived tumor model. While all control animals showed rapid tumor growth by bioluminescent imaging (BLI) and succumbed to disease within five weeks, all treated mice survived for the duration of the study. After P-BCMA-ALLO1 administration, tumor burden was rapidly reduced to the limit of detection by BLI within 7-10 days. Lastly, several occurrences of tumor relapse were observed in some of these animals, but were subsequently controlled without re-administration of the product. Altogether, these data demonstrate that P-BCMA-ALLO1 exhibited significantly reduced alloreactivity yet retained potent effector function, suggesting its safe and effective use for treating any MM patient. DisclosuresWang:Poseida Therapuetics: Employment. Barnett:Poseida Therapeutics, Inc.: Employment. Martin:Poseida Therapeutics, Inc.: Employment. Hermanson:Poseida Therapeutics: Employment. Li:Poseida Therapuetics: Employment. Smith:Poseida Therapeutics, Inc.: Employment. Wu:Poseida Therapuetics: Employment. Cranert:Poseida Therapuetics: Employment. Rengarajan:Poseida Therapeutics, Inc.: Employment. Tan:Poseida Therapeutics, Inc.: Employment. Tong:Poseida Therapuetics: Employment. Codde:Poseida Therapeutics, Inc.: Employment. Down:Poseida Therapeutics, Inc.: Employment. Ostertag:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Shedlock:Poseida Therapeutics, Inc.: Employment.

Influence of solution phase environmental heterogeneity and fluctuations on vibronic spectra: Perylene diimide molecular chromophore complexes in solution.

Ensembles of ab initio parameterized Frenkel-exciton model Hamiltonians for different perylene diimide dimer systems are used, together with various dissipative quantum dynamics approaches, to study the influence of the solvation environment and fluctuations in chromophore relative orientation and packing on the vibronic spectra of two different dimer systems: a π-stacked dimer in aqueous solution in which the relative chromophore geometry is strongly confined by a phosphate bridge and a side-by-side dimer in dichloromethane involving a more flexible alkyne bridge that allows quasi-free rotation of the chromophores relative to one another. These entirely first-principles calculations are found to accurately reproduce the main features of the experimental absorption spectra, providing a detailed mechanistic understanding of how the structural fluctuations and environmental interactions influence the vibronic dynamics and spectroscopy of solutions of these multi-chromophore complexes.

Role of dimeric gemini surfactant system on kinetic study of alanine amino acid with ninhydrin reaction

Role of dimeric gemini surfactant system on kinetic study of alanine amino acid with ninhydrin reaction

Efficient Four-Component Dirac-Coulomb-Gaunt Hartree-Fock in the Pauli Spinor Representation.

Four-component Dirac-Hartree-Fock is an accurate mean-field method for treating molecular systems where relativistic effects are important. However, the computational cost and complexity of the two-electron interaction make this method less common, even though we can consider the Dirac-Hartree-Fock Hamiltonian the "ground truth" of the electronic structure, barring explicit quantum electrodynamical effects. Being able to calculate these effects is then vital to the design of lower scaling methods for accurate predictions in computational spectroscopy and properties of heavy element complexes that must include relativistic effects for even qualitative accuracy. In this work, we present a Pauli quaternion formalism of maximal component and spin separation for computing the Dirac-Coulomb-Gaunt Hartree-Fock ground state, with a minimal floating point operation count algorithm. This approach also allows one to explicitly separate different spin physics from the two-body interactions, such as spin-free, spin-orbit, and spin-spin contributions. Additionally, we use this formalism to examine relativistic trends in the periodic table and analyze the basis set dependence of atomic gold and gold dimer systems.

Optical Parametric Oscillation with Ultra-Low Power Threshold in a Dimer of Active-Passive Cavities

Optical parametric oscillation can convert the input laser into a couple of coherent optical output with signal and idler frequencies. It is an important method for the realization of the broadband middle infrared tunable lasers. The optical parametric oscillation process depends on the ultra-fast non-linear response of matter to light with a certain pump power. Therefore, reducing the pump threshold of the optical parametric oscillation process and improving the energy conversion efficiency are of great significance to the study of non-linear optics. In this paper, we construct a dimer system that couples a passive non-linear resonator to an active resonator. Based on the dimer system, an ultra-low threshold optical parametric oscillation generation method is proposed. By coupling the gain pump mode, the non-linear effect is effectively enhanced, the pump power threshold is reduced to half of when there is no gain, and the energy conversion efficiency is increased. We believe this research provides a feasible method for a low-threshold tunable and easy-to-integrate optical parametric oscillation laser source.

Optogenetic Control of the Canonical Wnt Signaling Pathway During Xenopus laevis Embryonic Development

Optogenetics uses light-inducible protein-protein interactions to precisely control the timing, localization, and intensity of signaling activity. The precise spatial and temporal resolution of this emerging technology has proven extremely attractive to the study of embryonic development, a program faithfully replicated to form the same organism from a single cell. We have previously performed a comparative study for optogenetic activation of receptor tyrosine kinases, where we found that the cytoplasm-to-membrane translocation-based optogenetic systems outperform the membrane-anchored dimerization systems in activating the receptor tyrosine kinase signaling in live Xenopus embryos. Here, we determine if this engineering strategy can be generalized to other signaling pathways involving membrane-bound receptors. As a proof of concept, we demonstrate that the cytoplasm-to-membrane translocation of the low-density lipoprotein receptor-related protein-6 (LRP6), a membrane-bound coreceptor for the canonical Wnt pathway, triggers Wnt activity. Optogenetic activation of LRP6 leads to axis duplication in developing Xenopus embryos, indicating that the cytoplasm-to-membrane translocation of the membrane-bound receptor could be a generalizable strategy for the construction of optogenetic systems.

Functional assessment of PIP 2 regulation of ENaC using an optogenetic dimerization assay

The epithelial Na+ channel (ENaC) plays a key role in Na+ transport in epithelial linings to include the lung, colon and kidney. In the distal kidney tubules, ENaC regulates Na+ reabsorption and blood volume. Thus, dysfunctions in signaling pathways regulating ENaC activity are linked to hypertension or hypotension. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a target of the G protein coupled receptor P2Y2 pathway, and is necessary for the proper function of ENaC. This nonvoltage-gated trimeric channel is comprised of α, β, and γ subunits. We recently described two intracellular PIP2 binding sites on the N termini of β-, and γ-ENaC, with moderate μM affinity. Here, we report the functional effects on ENaC containing a combination of mutations to those PIP2 binding sites, by controlled depletion of PIP2. We used a CIBN/CRY2-5-ptase optogenetic dimerization system to deplete PIP2 levels in HEK293 cells transiently expressing wild type (wt) ENaC or the mutant ENaC constructs. CoroNa Green, a fluorescent Na+ indicator, was used to monitor ENaC activity by tracking the relative intracellular Na+ levels. Upon optogenetic-controlled depletion of PIP2, Na+ levels decreased in cells expressing wt ENaC. Mutations to the PIP2 sites of ENaC were expected to have no change in Na+ levels upon PIP2 depletion due to the disruption of PIP2 binding. As a control, mutations to non-PIP2 binding sites were included, and were expected to have decreased Na+ levels similar to wt ENaC. Interestingly, mutation of each independent PIP2 site resulted in only a small decrease of intracellular Na+, compared to wt ENaC. However, mutations throughout the entire N-terminus of β-ENaC, including the PIP2 binding site, resulted in a significant increase of Na+ upon PIP2 depletion. These data suggest that the residues surrounding the PIP2 binding sites play a significant role in the affinity of PIP2 for ENaC. The role of these other domains in PIP2 binding is still under investigation.

Mechanism for Synchronization of Charge Oscillations in Dimer Lattices

We discuss the mechanism and the conditions for the appearance of synchronized charge oscillations which have been observed experimentally and theoretically after strong photoexcitation of dimerized systems. In the Hubbard model with on-site repulsion, the Bloch equations for a wave-number-dependent pseudospin -- whose components describe the charge-density difference, current density, and bond density between the two sublattices -- involve an alternatingly tilted pseudomagnetic field, which assists the synchronization of pseudospins with different wave numbers, irrespective of the initial condition. This fact is numerically confirmed by the dynamics in finite lattices based on the exact diagonalization method. In the presence of nearest-neighbor repulsion, however, the synchronization can be hindered by excitons. Therefore, the excitation of a sufficiently large density of free electron-hole pairs, but low density of excitons, is needed to achieve synchronization.

Optimized iLID Membrane Anchors for Local Optogenetic Protein Recruitment.

Optogenetic protein dimerization systems are powerful tools to investigate the biochemical networks that cells use to make decisions and coordinate their activities. These tools, including the improved Light-Inducible Dimer (iLID) system, offer the ability to selectively recruit components to subcellular locations, such as micron-scale regions of the plasma membrane. In this way, the role of individual proteins within signaling networks can be examined with high spatiotemporal resolution. Currently, consistent recruitment is limited by heterogeneous optogenetic component expression, and spatial precision is diminished by protein diffusion, especially over long time scales. Here, we address these challenges within the iLID system with alternative membrane anchoring domains and fusion configurations. Using live cell imaging and mathematical modeling, we demonstrate that the anchoring strategy affects both component expression and diffusion, which in turn impact recruitment strength, kinetics, and spatial dynamics. Compared to the commonly used C-terminal iLID fusion, fusion proteins with large N-terminal anchors show stronger local recruitment, slower diffusion of recruited components, efficient recruitment over wider gene expression ranges, and improved spatial control over signaling outputs. We also define guidelines for component expression regimes for optimal recruitment for both cell-wide and subcellular recruitment strategies. Our findings highlight key sources of imprecision within light-inducible dimer systems and provide tools that allow greater control of subcellular protein localization across diverse cell biological applications.

Atomistic Modeling of Peptide Aggregation and β-Sheet Structuring in Corn Zein for Viscoelasticity.

The structure-function relationships of plant-based proteins that give rise to desirable texture attributes in order to mimic meat products are generally unknown. In particular, it is not clear how to engineer viscoelasticity to impart cohesiveness and proper mouthfeel; however, it is known that intermolecular β-sheet structures have the potential to enhance the viscoelastic property. Here, we investigated the propensity of selected peptide segments within common corn α-zein variants to maintain stable aggregates and β-sheet structures. Simulations on dimer systems showed that stability was influenced by the initial orientation and the presence of contiguous small hydrophobic residues. Simulations using eight-peptide β-sheet oligomers revealed that peptide sequences without proline had higher levels of β-sheet structuring. Additionally, we identified that sequences with a dimer hydrogen-bonding density of >22% tended to have a larger percent β-sheet conformation. These results contribute to understanding how the viscoelasticity of zein can be increased for use in plant-based meat analogues.

Chemical Dimerization-Induced Protein Condensates on Telomeres.

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a chemically-induced protein dimerization system to create condensates on telomeres. The chemical dimerizer consists of two linked ligands that can each bind to a protein: Halo ligand to Halo-enzyme and trimethoprim (TMP) to E. coli dihydrofolate reductase (eDHFR), respectively. Fusion of Halo enzyme to a telomere protein anchors dimerizers to telomeres through covalent Halo ligand-enzyme binding. Binding of TMP to eDHFR recruits eDHFR-fused phase separating proteins to telomeres and induces condensate formation. Because TMP-eDHFR interaction is non-covalent, condensation can be reversed by using excess free TMP to compete with the dimerizer for eDHFR binding. An example of inducing promyelocytic leukemia (PML) nuclear body formation on telomeres and determining condensate growth, dissolution, localization and composition is shown. This method can be easily adapted to induce condensates at other genomic locations by fusing Halo to a protein that directly binds to the local chromatin or to dCas9 that is targeted to the genomic locus with a guide RNA. By offering the temporal resolution required for single cell live imaging while maintaining phase separation in a population of cells for biochemical assays, this method is suitable for probing both the formation and function of chromatin-associated condensates.

Luciferase Controlled Protein Interactions.

Protein trafficking and protein-protein interactions (PPIs) are central to regulatory processes in cells. Induced dimerization systems have been developed to control PPIs and regulate protein trafficking (localization) or interactions. Chemically induced dimerization (CID) has proven to be a robust approach to control protein interactions and localization. The most recent embodiment of this technology relies on CID conjugates that react with a self-labeling protein on one side and a photocaged ligand on the other side to provide spatiotemporal control of the interaction with the protein of interest. Advancing this technology further is limited by the light delivery problem and the phototoxicity of intense irradiation necessary to achieve photouncaging. Herein, we designed a novel chemically induced dimerization system that was triggered by bioluminescence, instead of external light. Protein dimerization showed fast kinetics and was validated by an induced change of localization of a target protein (to and from the nucleus or plasma membrane) upon trigger. The technology was used transiently to activate the phosphatidylinositol 3-kinase (PI3K)/mTOR pathway and measure the impact on lipid synthesis/metabolism, assessed by lipidomics.