Biology subjects

Mokhtari, D. A.

Publications and source records attributed to Mokhtari, D. A..

7 recordsLinked to original sources

bioRxiv2026

High-throughput biochemical phenotyping of SHP2 variants reveals molecular basis of diseases and allosteric drug inhibition

Interpreting clinical and functional consequences of genetic variants remains challenging due to limited quantitative biochemical data at scale. We applied high-throughput microfluidic enzyme kinetics to profile 190 clinical variants of SHP2, a protein tyrosine phosphatase linked to developmental disorders and cancers. Through >300,000 reaction progress curves, we derived kinetic and thermodynamic parameters quantifying variant effects on catalysis, autoinhibition, stability, phosphopeptide binding, and drug responses. This multidimensional dataset reveals that dysregulated autoinhibition, rather than altered stability or catalysis, predominantly determines SHP2-associated pathogenesis. Thermodynamic modeling reveals that clinical-stage allosteric inhibitors preferentially stabilize a previously underappreciated, partially active conformation over the fully inactive state, leading to variant-dependent drug responses. Our high-throughput biochemical framework establishes a general approach to decipher the biochemical logic connecting protein variants to clinical outcomes.

biochemistry↗

bioRxiv2025

Large field of view fluorescence imaging of microfluidic devices with a tandem-lens macroscope

Microfluidic devices enable high-throughput sample processing with remarkable parallelization and miniaturization. While fluorescence microscopy provides a convenient method for reading out signal from microfluidic assays, commercially-available microscopes impose a fundamental tradeoff between temporal resolution, spatial resolution, and numerical aperture (NA). Spatially tiled imaging enables high-resolution and high-NA imaging over a large area but reduces temporal resolution. Conversely, low magnification, low NA imaging captures large areas in one shot, but typically sacrifices spatial resolution and fluorescence sensitivity. To address this, we introduce an automated transfluorescence tandem-macro-lens optomechanical system (macroscope) capable of sensitive, multi-channel fluorescence imaging over a very large field of view (34 mm diameter, 740 mm2), with resolution determined by the sensor pixel size. We demonstrate bright-field resolution of low-micron features and detection of low-to mid-nanomolar concentrations of common fluorophores within microfluidic device channels. To demonstrate the utility of this macroscope, we image enzyme turnover within valved microfluidic devices (the HT-MEK system, for High-Throughput Microfluidic Enzyme Kinetics) and achieve >50-fold increased temporal resolution over common commercial instruments while maintaining high sensitivity. This macroscope imaging solution costs substantially less than commercially available alternatives, providing a powerful new imaging approach for microfluidic applications requiring sensitive and rapid wide-field fluorescence imaging.

bioengineering↗

bioRxiv2022

High-throughput enzymology reveals mutations throughout a phosphatase that decouple catalysis and transition state analog affinity

Using High-Throughput Microfluidic Enzyme Kinetics (HT-MEK), we measured over 9,000 inhibition curves detailing impacts of 1,004 single-site mutations throughout the Alkaline Phosphatase PafA on binding affinity for two transition state analogs (TSAs), vanadate and tungstate. As predicted by catalytic models invoking transition state complementary, mutations to active site and active site-contacting residues had highly similar impacts on catalysis and TSA binding. Unexpectedly, most mutations to more distal residues which reduced catalysis had little or no impact on TSA binding and many even increased affinity for tungstate. These disparate effects are accounted for by a model in which distal mutations alter the enzymes conformational landscape and increase occupancy of microstates that are catalytically less effective but better able to accommodate larger transition state analogs. In support of this model, glycine substitutions (rather than valine) were more likely to increase tungstate affinity, presumably due to increased conformational flexibility and increased occupancy of previously disfavored microstates. These results indicate that residues throughout an enzyme provide specificity for the transition state and discriminate against analogs that are larger only by tenths of an [A]ngstrom. Thus, engineering enzymes that rival the most powerful natural enzymes will likely require consideration not just of residues in and around the active site, but also of more distal residues that shape the enzymes conformational landscape and finetune the active site. In addition, the extensive functional communication between the active site and remote residues may provide interconnections needed for allostery and make allostery a highly evolvable trait. Significance StatementTransition state analogs (TSAs) resemble fleeting high-energy transition states and have been used to inhibit enzymes in nature and medicine, to learn about enzyme active site features, and to design and select new enzymes. While TSAs mimic transition states, they differ from actual TSs, and we exploit these differences here. Systematic TSA affinity measurements for 1,004 mutants of PafA (a model phosphatase enzyme) revealed effects in and around the active site that mirror their effects on catalysis, but TSA-binding and catalytic effects diverge more distally. These observations suggest that residues throughout an enzyme adjust its conformational landscape on the tenth-[A]ngstrom scale to optimize the active site for catalysis, rendering allostery more evolvable in nature but likely complicating enzyme design.

biochemistry↗

bioRxiv2022

A microwell platform for high-throughput longitudinal phenotyping and selective retrieval of organoids

Organoids are powerful experimental models for studying the ontogeny and progression of diseases including cancer. Organoids are conventionally cultured in bulk using an extracellular matrix mimic. However, organoids in bulk culture physically overlap, making it impossible to track the growth of individual organoids over time in high throughput. Moreover, local spatial variations in bulk matrix properties make it difficult to assess whether observed phenotypic heterogeneity between organoids results from intrinsic cell differences or microenvironment variability. Here, we developed a microwell-based method that enables high-throughput quantification of image-based parameters for organoids grown from single cells, which can be retrieved from their microwells for sequencing and molecular profiling. Coupled with a deep-learning image processing pipeline, we characterized phenotypic traits including growth rates, cellular movement, and apical-basal polarity in two CRISPR-engineered human gastric organoid models, identifying genomic changes associated with increased growth rate and changes in accessibility and expression correlated with apical-basal polarity.

bioengineering↗

bioRxiv2021

Damaged goods? Evaluating the impact of X-ray damage on conformational heterogeneity in room temperature and cryo-cooled protein crystals

X-ray crystallography is a cornerstone of biochemistry. Traditional freezing of protein crystals to cryo-temperatures mitigates X-ray damage and facilitates crystal handling but provides an incomplete window into the ensemble of conformations at the heart of protein function and energetics. Room temperature (RT) X-ray crystallography provides more extensive ensemble information, and recent developments allow conformational heterogeneity, the experimental manifestation of ensembles, to be extracted from single crystal data. However, high sensitivity to X-ray damage at RT raises concerns about data reliability. To systematically address this critical question, we obtained increasingly X-ray-damaged high-resolution datasets (1.02-1.52 [A]) from single thaumatin, proteinase K, and lysozyme crystals. Heterogeneity analyses indicated a modest increase in conformational disorder with X-ray damage. Nevertheless, these effects do not alter overall conclusions and can be minimized by limiting the extent of X-ray damage or eliminated by extrapolation to obtain heterogeneity information free from X-ray damage effects. To compare these effects to damage at cryo temperature and to learn more about damage and heterogeneity in cryo-cooled crystals, we carried out an analogous analysis of increasingly damaged proteinase K cryo datasets (0.9-1.16 [A]). We found X-ray damage-associated heterogeneity changes that were not observed at RT. This observation and the scarcity of reported X-ray doses and damage extent render it difficult to distinguish real from artifactual conformations, including those occurring as a function of temperature. The ability to aquire reliable heterogeneity information from single crystals at RT provides strong motivation for further development and routine implementation of RT X-ray crystallography to obtain conformational ensemble information. SignificanceX-ray crystallography has allowed biologists to visualize the proteins that carry out complex biological processes and has provided powerful insights into how these molecules function. Our next level of understanding requires information about the ensemble of conformations that is at the heart of protein function and energetics. Prior results have shown that room temperature (RT) X-ray crystallography provides extensive ensemble information, but are subject to extenstive X-ray damage. We found that ensemble information with little or no effects from X-ray damage can be collected at RT. We also found that damage effects may be more prevalent than recognized in structures obtained under current standard cryogenic conditions. RT X-ray crystallography can be routinely implemented to obtain needed information about conformational ensembles.

biophysics↗

bioRxiv2020

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK, a microfluidic platform for high-throughput expression, purification, and characterization of >1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA, we performed >670,000 reactions to determine >5000 kinetic and physical constants for multiple substrates and inhibitors. These constants allowed us to uncover extensive kinetic partitioning to a misfolded state and isolate catalytic effects, revealing spatially contiguous "regions" of residues linked to particular aspects of function. These regions included active-site proximal residues but also extended to the enzyme surface, providing a map of underlying architecture that could not be derived from existing approaches. HT-MEK, using direct and coupled fluorescent assays, has future applications to a wide variety of problems ranging from understanding molecular mechanisms to medicine to engineering and design. One Sentence SummaryHT-MEK, a microfluidic platform for high-throughput, quantitative biochemistry, reveals enzyme architectures shaping function.

biochemistry↗

bioRxiv2020

High-throughput binding affinity measurements for mutations spanning a transcription factor-DNA interface reveal affinity and specificity determinants

Transcription factors (TFs) bind regulatory DNA to control gene expression, and mutations to either TFs or DNA can alter binding affinities to rewire regulatory networks and drive phenotypic variation. While studies have profiled energetic effects of DNA mutations extensively, we lack similar information for TF variants. Here, we present STAMMP (Simultaneous Transcription Factor Affinity Measurements via Microfluidic Protein Arrays), a high-throughput microfluidic platform enabling quantitative characterization of hundreds of TF variants simultaneously. Measured affinities for [~]210 mutants of a model yeast TF (Pho4) interacting with 9 oligonucleotides (>1,800 Kds) reveal that many combinations of mutations to poorly conserved TF residues and nucleotides flanking the core binding site alter but preserve physiological binding, providing a mechanism for mutations in cis and trans to rewire networks without insurmountable evolutionary penalties. Moreover, biochemical double-mutant cycles across the TF-DNA interface reveal molecular mechanisms driving recognition, linking sequence to function.

biophysics↗