Biology subjects

Yoon, T.-Y.

Publications and source records attributed to Yoon, T.-Y..

7 recordsLinked to original sources

bioRxiv2026

Landscape-scale navigation unlocks antibody CDR structural logic for AI-guided rescue and therapeutic optimization

While AI offers transformative potential for therapeutic antibody design, the lack of ground-truth data fundamentally constrains our ability to model the epistatic topology of fitness landscapes. Here, we establish a high-throughput workflow to characterize tens of thousands of antibody variants per week with gold-standard biophysical precision. By combinatorially assembling functional variants from deep mutational scanning, we charted antibody fitness landscapes comprising over 17,000 data points, which revealed an extremely rugged, non-navigable epistatic topology. Yet, navigating at this unprecedented scale enabled the discovery of rare peak clusters exhibiting simultaneous enhancements in affinity and productivity. Strikingly, ProteinMPNN predicted the CDR-dependent productivity landscape with remarkable accuracy, suggesting that sequence-structure compatibility within CDRs gates cellular productivity. This insight enabled a structure-guided rescue strategy combining AlphaFold3 and ProteinMPNN, which successfully restored the cellular productivity of high-affinity, low-productivity clones via single amino acid substitutions. Two elite variants drawn directly from peak clusters further demonstrated 20- to 100-fold in vivo efficacy gains in a murine psoriasis model. Our findings establish CDR structural fitness as a fundamental determinant of antibody cellular productivity and validate landscape-scale navigation as a powerful framework for therapeutic antibody optimization.

bioengineering↗

bioRxiv2026

Single-molecule dissection of CFTR folding defects and pharmacological rescue

Cystic fibrosis is a lethal genetic disorder caused by misfolding of the CFTR protein, most commonly due to the {Delta}F508 mutation. Despite extensive study, CFTRs folding process has remained inaccessible to direct observation. Here, we apply single-molecule magnetic tweezers to resolve the complete folding trajectories of wild-type and {Delta}F508 CFTR with near-amino acid resolution. We find that CFTR follows a hierarchical, template-guided folding pathway in which N-terminal domains scaffold downstream folding. This mechanism tightly couples the free energy states of intermediates, allowing {Delta}F508-induced instability to propagate across the folding pathway. Pharmacological correctors, in synergy with ATP, reshape the entire folding energy landscape by catalyzing transitions rather than simply stabilizing end states. These long-range, allosteric effects reveal a folding-embedded regulatory network. Our work provides a quantitative framework for mapping multidomain protein folding and therapeutic rescue, offering a broadly applicable strategy for interrogating rare mutations and accelerating structure-based drug discovery. Significance StatementMisfolding of CFTR underlies cystic fibrosis, and its complex, multidomain architecture makes it an ideal model for understanding how membrane proteins fold and how small molecules can restore native structure. Using single-molecule magnetic tweezers, we reveal how local instabilities propagate through CFTRs folding pathway and show that pharmacological correctors act by catalyzing specific folding transitions in addition to stabilizing the native fold. These insights establish CFTR as a paradigm for dissecting folding mechanisms in large membrane proteins and for developing general strategies to correct misfolding across diverse human diseases.

biophysics↗

bioRxiv2024

Mapping and Engineering Antibody-Antigen Interaction Landscapes for Systematic Affinity Enhancement

Antibodies, crucial in adaptive immunity, recognize antigens through specific interactions facilitated by Complementarity Determining Regions (CDRs), diversified via Variable-Diversity-Joining (VDJ) recombination. Traditional antibody development, limited by the scope of animal models and phage display libraries, captures a fraction of the potential antibody-antigen interactions. This underscores a gap in understanding antibody specificity and the relationship between antibody sequence and binding affinity. Here we introduce an approach using the Single-Protein Interaction Detection (SPID) platform, repurposed to systematically map local landscapes of antibody-antigen interactions with unprecedented depth and speed, aiming to rival the precision of methods like Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI) while significantly boosting throughput. By editing CDR sequences and measuring effects on dissociation constants, we elucidated pathways for optimizing antibody affinity, enhancing predictive models for interactions. Our findings demonstrate the capability of the SPID platform to characterize thousands of variants weekly, offering a deeper insight into antibody-antigen interactions and advancing antibody development with finely-tuned affinities.

bioengineering↗

bioRxiv2024

Profiling multi-body interactions of BCL2 with single-molecule co-immunoprecipitation reveals the molecular mechanism of ABT-199 resistance

A capability to characterize protein-protein interactions (PPIs) in a quantitative manner with an increased speed would form a technical basis for accelerating drug discovery targeting the PPI network. We here used the single-molecule pull-down and co-IP platform to examine PPI between BCL2 and BH3-only proteins in crude extract environments. We focused on how the PPI strengths changed with single-point BCL2 mutations found in relapsed chronic lymphocytic leukemia showing ABT-199 resistance, where we took a mix-and-match approach to examine various pairs of baits and preys while titrating their concentrations. This allowed us to examine total 21 PPI reactions and 420 data points, forming a high-resolution large data set of the dissociation constants (Kd) and the drug inhibitory constants (Ki). Our data suggest that the different BCL2 mutants take different routes to acquire resistance to ABT-199, demonstrating how large-scale, quantitative PPI data sets reveal insights into the evolving dynamics of PPI networks.

biochemistry↗

bioRxiv2023

Predicting the efficacy of BH3 mimetics through the profiling of multiple protein complexes

BH3 mimetics are protein-protein interaction (PPI) inhibitors that saturate anti-apoptotic proteins in the BCL2 family to induce apoptosis in cancer cells, a prominent example of protein complex-targeting therapies. Despite the remarkable success of the BH3 mimetic ABT-199 in treating hematological malignancies, only a fraction of patients respond to ABT-199 and eventually develop resistance, necessitating the predictive biomarkers for both initial responses and resistance development. We here used the single-molecule pull-down and co-immunoprecipitation platform to quantify more than 20 different types of PPI complexes using [~]1.2x106 cells in total, revealing the rewired status of the BCL2 family PPI network. By comparing the obtained multi-dimensional data with BH3 mimetic efficacies determined ex vivo, we constructed an analysis model for ABT-199 efficacy that designates the BCL2-BAX and BCLxL-BAK complexes as the primary mediators of drug effectiveness and resistance. We then applied this model to assist in therapeutic decision-making for acute myeloid leukemia patients in a prospective manner. Our work demonstrates a capability for the extensive characterization of PPI complexes in clinical specimens, potentially opening a new avenue of precision medicine for protein complex-targeting therapies.

molecular biology↗

bioRxiv2022

Exocyst stimulates each step of exocytic SNARE complex assembly and vesicle fusion

The exocyst is a large multisubunit tethering complex essential for targeting and fusion of secretory vesicles in eukaryotic cells. Although the assembled exocyst complex is proposed to tether vesicles to the plasma membrane and activate the SNARE proteins for membrane fusion, only little is known about the key biochemical steps that exocyst stimulates in the course of SNARE complex assembly, a critical question defining the essential molecular role of the exocyst complex. Here, we use a combination of single molecule and bulk fluorescence assays with purified octameric yeast exocyst complexes to examine the role of exocyst in a reconstituted SNARE assembly and fusion system. We show that the exocyst complex simulates multiple steps spanning from SNARE protein activation to ternary complex assembly, rather than affecting only a specific subset of steps. We also observed that the exocyst has important downstream roles in driving membrane fusion, up to full content mixing of vesicle lumens. Our results suggest that the exocyst complex provides extensive chaperoning functions for the entire process of SNARE complex assembly, presumably using its multi-faceted structure provided by the eight subunits.

molecular biology↗

bioRxiv2020

Encoding multiple virtual signals in DNA barcodes with single-molecule FRET

DNA barcoding provides a way to label a huge number of different biological molecules using the extreme programmability in DNA sequence synthesis. Fluorescence imaging is an easy-to-access method to detect individual DNA barcodes, which can be scaled up to a massively high-throughput format. Large overlaps between emission spectra of fluorescence dyes, however, severely limit the numbers of DNA barcodes-and thus its signal space-that can be detected in a simultaneous manner. We here demonstrate the use of single-molecule fluorescence resonance energy transfer (FRET) to encode virtual signals in DNA barcodes using conventional two-color fluorescence microscopy. By optimizing imaging and biochemistry conditions for weak hybridization events for DNA barcodes, we markedly enhanced accuracy in our determination of the efficiency by which single-molecule FRET occurred. This allowed us to unambiguously differentiate six DNA barcodes exhibiting different FRET values without involving probe sequence exchanges. Our method can be directly incorporated with previous DNA barcode techniques, and may thus be widely adopted to expand the signal space of the DNA barcode techniques.

biophysics↗