Biology subjects

Sunden, F.

Publications and source records attributed to Sunden, F..

5 recordsLinked to original sources

bioRxiv2026

Zero-shot design of a de novo metalloenzyme

The de novo design of enzymes remains a central challenge, requiring consideration of catalytic mechanism and optimization across biochemical and biophysical criteria. Here we present dEVA (design by EVolutionary Algorithm), a multi-objective protein design framework built on principles drawn from evolutionary biology. We apply dEVA to the zero-shot, de novo design of metalloenzymes by optimizing the coordination sphere of catalytic metals. We characterize a bizinc metalloenzyme that exhibits promiscuous hydrolytic activity towards both phosphomonoesters and phosphodiesters. This design achieves a rate enhancement ((kcat/KM)/kw) up to 3 x 1013, comparable to characterized natural phosphatases. dEVA offers a general and modular strategy for the programmable design of protein function without dependence on natural templates or evolutionary information.

biochemistry↗

bioRxiv2025

Quantifying protein unfolding kinetics with a high-throughput microfluidic platform

Even after folding, proteins transiently sample unfolded or partially unfolded intermediates, and these species are often at risk of irreversible alteration (e.g. via proteolysis, aggregation, or post-translational modification). Kinetic stability, in addition to thermodynamic stability, can directly impact protein lifetime, abundance, and the formation of alternative, sometimes disruptive states. However, we have very few measurements of protein unfolding rates or how mutations alter these rates, largely due to technical challenges associated with their measurement. To address this need, we developed SPARKfold (Simultaneous Proteolysis Assay Revealing Kinetics of Folding), a microfluidic platform to express, purify, and measure unfolding rate constants for >1000 protein variants in parallel via on-chip native proteolysis. To demonstrate the power and potential of SPARKfold, we determined unfolding rate constants for 1,104 protein samples in parallel. We built a library of 31 dihydrofolate reductase (DHFR) orthologs with up to 78 chamber replicates per variant to provide the statistical power required to evaluate the systems ability to resolve subtle effects. SPARKfold rate constants for 5 constructs agreed with those obtained using traditional techniques across a 150-fold range, validating the accuracy of the technique. Comparisons of mutant kinetic effects via SPARKfold with previously published measurements impacts on folding thermodynamics provided information about the folding transition state and pathways via {varphi} analysis. Overall, SPARKfold enables rapid characterization of protein variants to dissect the nature of the unfolding transition state. In future work, SPARKfold can reveal mutations that drive misfolding and aggregation and enable rational design of kinetically hyperstable variants for industrial use in harsh environments.

biochemistry↗

bioRxiv2024

Hydrophilic/ Omniphobic droplet arrays for high-throughput and quantitative enzymology

Engineered enzymes with enhanced or novel functions are specific catalysts with wide-ranging applications in industry and medicine. Here, we introduce Droplet Array Microfluidic Enzyme Kinetics (DA-MEK), a high-throughput enzyme screening platform that combines water-in-air droplet microarrays formed on patterned superhydrophilic/omniphobic surfaces with cell-free protein synthesis to enable cost-effective expression and quantitative kinetic characterization of enzyme variants. By printing DNA templates encoding enzyme variants onto hydrophilic spots, stamping slides to add cell-free expression mix, and imaging the resulting arrays, we demonstrate reproducible expression of hundreds of enzyme variants per slide. Subsequent stamping of fluorogenic substrates and time-lapse imaging allows determination of Michaelis-Menten parameters for each variant, with measured catalytic efficiencies spanning 5 orders of magnitude and agreeing well with values obtained via traditional microtiter plate assays. DA-MEK consumes orders of magnitude less reagents than plate-based assays while providing accurate and detailed kinetic information for both beneficial and deleterious mutations. In future work, we anticipate DA-MEK will provide a powerful and versatile platform to accelerate enzyme engineering and enable screening of large variant libraries under diverse conditions.

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↗

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↗