Biology subjects

Ha, J.-H.

Publications and source records attributed to Ha, J.-H..

8 recordsLinked to original sources

bioRxiv2025

Next Generation of StayGold-based Adaptable Turn-On Maturation (ATOM) Sensors Targeting PSD95, Gephyrin, and HOMER1 Proteins

Genetically encoded biosensors that have the capacity to detect intracellular molecules have proven to be invaluable tools in biology. However, development of biosensors with generalizable binding domains for detecting arbitrary targets of choice is still in its early stages. We previously introduced ATOM biosensor technology in which adaptable monobody and nanobody binding domains are employed for molecular recognition, and biosensor turn-on is achieved by coupling target binding to activation of the attached fluorescent protein by means of conformational change. In this work, we extend the ATOM mechanism to the newly identified, highly photostable fluorescent proteins mStayGold and mBaoJin. We validated the ATOM sensors using three neuronal targets: PSD95, Gephyrin, and HOMER1. The sensors exhibited turn-on ratios of over 100-fold with high specificity. Compared to existing methods for detecting these proteins, ATOM sensors demonstrated improved dynamic range, lower background, and a higher turn-on. The results highlight the adaptable nature of the ATOM mechanism and demonstrate that it allows for development of biosensors with various colors and photophysical characteristics.

synthetic biology↗

bioRxiv2023

Mimicking Kidney Flow Shear Efficiently Induces Aggregation of LECT2, a Protein Involved in Renal Amyloidosis

Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Homozygosity of the I40V LECT2 mutation is believed to be necessary but not sufficient for the disease. Previous studies established that LECT2 fibrillogenesis is greatly accelerated by loss of its single bound zinc ion and stirring or shaking. These forms of agitation are often used to facilitate protein aggregation, but they create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress--shear generated by fluid flow through a network of artery and capillary-sized channels--drives LECT2 fibrillogenesis. To mimic blood flow through the human kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 m in width. Flow shear was particularly pronounced at the branch points and in the smallest capillaries, and this induced LECT2 aggregation much more efficiently than conventional shaking methods. EM images suggested the resulting fibril structures were different in the two conditions. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both size and density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering the mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.

biochemistry↗

bioRxiv2023

Adaptable, Turn-On Monobody (ATOM) Fluorescent Biosensors for Multiplexed Detection in Cells

A grand challenge in biosensor design is to develop a single molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Conceptually, this can be achieved by fusing a small, antibody-like binding domain to a fluorescent protein in such a way that target binding activates fluorescence. Although this design is simple to envision, its execution is not obvious. Here, we created a family of adaptable, turn-on monobody (ATOM) biosensors consisting of a monobody, circularly permuted at one of two positions, inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells co-expressing cyan, yellow, and red ATOM sensors detected the biosensor targets (WDR5, SH2, and hRAS proteins) that were localized to the nucleus, cytoplasm, and plasma membrane, respectively, with high specificity. ER- and mitochondria-localized ATOM sensors also detected ligands that were targeted to those organelles. Fluorescence activation involved ligand-dependent chromophore maturation with fluorescence turn-on ratios of >20-fold in cells and up to 100-fold in vitro. The sensing mechanism was validated with three arbitrarily chosen monobodies inserted into jellyfish as well as anemone lineages of fluorescent proteins, suggesting that ATOM sensors with different binding specificities and additional colors can be generated relatively quickly.

synthetic biology↗

bioRxiv2022

A Generalizable Nanopore Sensor for Highly Specific Protein Detection at Single-Molecule Precision

Protein detection and biomarker profiling have wide-ranging implications in many areas of basic research and molecular diagnostics. Substantial progress has been made in protein analytics using nanopores and the resistive-pulse technique. Yet, a long-standing challenge is implementing specific binding interfaces for detecting proteins without the steric hindrance of the pore interior. To overcome this technological difficulty, we formulate a new class of sensing elements made of a programmable antibody-mimetic binder fused to a monomeric protein nanopore. This way, such a modular design significantly expands the utility of nanopore sensors to numerous proteins while preserving their architecture, specificity, and sensitivity. We prove the power of this approach by developing and validating nanopore sensors for protein analytes that drastically vary in size, charge, and structural complexity. These analytes produce unique electrical signatures that depend on their identity and quantity and the binder-analyte assembly at the nanopore tip. From a practical point of view, our sensors unambiguously probe protein recognition events without the necessity of using any additional exogenous tag. The outcomes of this work will impact biomedical diagnostics by providing a fundamental basis and tools for protein biomarker detection in biofluids.

biophysics↗

bioRxiv2022

An adaptable, monobody-based biosensor scaffold with FRET output

Protein-based fluorescent biosensors are powerful tools for analyte recognition in vitro and in cells. Numerous proteinaceous binding scaffolds have been developed that recognize ligands with affinity and specificity comparable to those of conventional antibodies, but are smaller, readily overexpressed, and more amenable to engineering. Like antibodies, these binding domains are useful as recognition modules in protein switches and biosensors, but they are not capable of reporting on the binding event by themselves. Here, we engineer a small binding scaffold--a consensus-designed fibronectin 3 monobody--such that it undergoes a conformational change upon ligand binding. This change is detected by Forster resonance energy transfer using chemical dyes or cyan and yellow fluorescent proteins as donor/acceptor groups. By grafting substrate recognition residues from different monobodies onto this scaffold, we create fluorescent biosensors for c-Abl Src homology 2 (SH2) domain, WD40-repeat protein 5 (WDR5), small ubiquitin-like modifier-1 (SUMO), and h-Ras. The biosensors bind their cognate ligands reversibly, with affinities consistent with those of the parent monobodies, and with half times of seconds to minutes. This design serves as generalizable platform for creating a genetically-encoded, ratiometric biosensors by swapping binding residues from known monobodies, with minimal modification.

biochemistry↗

bioRxiv2022

Ultramicrobacteria in various fermented cabbages

Little is known about the ultramicrobial communities of foods. Several bacteria, including pathogenic species, can form ultramicrobial communities when exposed to harsh conditions, making their detection via conventional culture techniques difficult. This study aimed to explore ultramicrobial communities within the microbial communities of fermented cabbage products, such as kimchi, sauerkraut, and suancai, which are produced through specific manufacturing methods based on the laws and culture of respective regions. We used single-molecule real-time sequencing with tangential flow filtration for fermented cabbages after pre-filtration and transmission electron microscopy to confirm the identity of ultramicrobacteria (UMB).To the best of our knowledge, this is the first study to identify the differences between ultramicrobial communities and microbial communities of fermented cabbages. Although the size of the ultramicrobial communities was smaller than that of the latter, their diversity was not lower. In addition, some UMB underwent cell shrinkage due to unfavorable environments, while others maintained their small size regardless. Major pathogens were not detected in the ultramicrobial communities of fermented cabbages. Nevertheless, several suspicious strains were detected. Our method can be used to screen food materials for the presence of UMB undetectable via conventional methods. Ultramicrobial and microbial communities were efficiently separated using tangential flow filtration and analyzed via single-molecule real-time sequencing. The ultramicrobial communities of fermented vegetables were different from conventional microbial communities. This study provides new insights into the ecology of UMB in foods.

microbiology↗

bioRxiv2021

Engineering A Fluorescent Protein Color Switch Using Entropy-driven Beta Strand Exchange

Protein conformational switches are widely used in biosensing. They are typically composed of an input domain (which binds a target ligand) fused to an output domain (which generates an optical readout). A central challenge in designing such switches is to develop mechanisms for coupling the input and output signals via conformational change. Here, we create a biosensor in which binding-induced folding of the input domain drives a conformational shift in the output domain that results in a 6-fold green-to-yellow ratiometric fluorescence change in vitro, and a 35-fold intensiometric fluorescence increase in cultured cells. The input domain consists of circularly permuted FK506 binding protein (cpFKBP) that folds upon binding its target ligand (FK506 or rapamycin). cpFKBP folding induces the output domain, an engineered GFP variant, to replace one of its {beta}-strands (containing T203 and specifying green fluorescence) with a duplicate {beta}-strand (containing Y203 and specifying yellow fluorescence) in an intramolecular exchange reaction. This mechanism employs the loop-closure entropy principle, embodied by folding of the partially disordered cpFKBP domain, to couple ligand binding to the GFP color shift. This proof-of-concept design has the advantages of full genetic encodability, ratiometric or intensiometric response, and potential for modularity. The latter attribute is enabled by circular permutation of the input domain.

biophysics↗

bioRxiv2020

Zinc shapes the folding landscape of p53 and establishes a new pathway for reactivating structurally diverse p53 mutants

Missense mutations in the DNA binding domain (DBD) of the p53 tumor suppressor contribute to approximately half of new cancer cases each year worldwide. A primary goal in cancer therapy is to develop drugs that rescue the transcription function of mutant p53. Here we present a thermodynamic model that quantifies and links the major pathways by which mutations inactivate p53. The model is constructed by measuring folding free energies, zinc dissociation constants, and DNA dissociation constants of 20 of the most common DBD mutations in the p53 database. We report here that DBD possesses two unusual properties----one of the highest zinc binding affinities of any eukaryotic protein and extreme instability in the absence of zinc--which are predicted to cause p53 to be poised on the edge of folding/unfolding in the cell, with a major determinant being the concentration of available zinc. Eighty percent of the mutations examined impair either thermodynamic stability, zinc binding affinity, or both. Using a combination of biophysical experiments, cell based assays, and murine cancer models, we demonstrate for the first time that a synthetic zinc metallochaperone not only rescues mutants with decreased zinc affinities, but also mutants that destabilize DBD without impairing zinc binding. The latter is a broad class of p53 mutants of which only one member (Y220C) has been successfully targeted by small molecules. The results suggest that zinc metallochaperones have the capability to treat 120,500 patients per year in the U.S. SUMMARYRestoring tumor suppressing function to mutant p53 has the capability of treating millions of new cancer patients worldwide each year. An important step toward this goal is to categorize the spectrum of mutations based on how they inactivate p53. This study finds that the majority of the most common tumorigenic mutations compromise p53s thermodynamic stability or its interaction with zinc, and demonstrates for the first time that members of both classes can be reactivated in cells by synthetic zinc metallochaperones. These results serve to stratify patients for potential zinc metallochaperone therapy.

biochemistry↗