Single-domain VHH antibodies are promising reagents for medical therapy. A conserved disulfide bond within the VHH framework region is known to be critical for thermal stability, however, no prior ...studies have investigated its influence on the stability of VHH antibody–antigen complexes under mechanical load. Here, we used single-molecule force spectroscopy to test the influence of a VHH domain’s conserved disulfide bond on the mechanical strength of the interaction with its antigen mCherry. We found that although removal of the disulfide bond through cysteine-to-alanine mutagenesis significantly lowered VHH domain denaturation temperature, it had no significant impact on the mechanical strength of the VHH:mCherry interaction with complex rupture occurring at ∼60 pN at 103–104 pN/sec regardless of disulfide bond state. These results demonstrate that mechanostable binding interactions can be built on molecular scaffolds that may be thermodynamically compromised at equilibrium.
Single‐molecule force spectroscopy (SMFS) is powerful for studying folding states and mechanical properties of proteins, however, it requires protein immobilization onto force‐transducing probes such ...as cantilevers or microbeads. A common immobilization method relies on coupling lysine residues to carboxylated surfaces using 1‐ethyl‐3‐(3‐dimethyl‐aminopropyl) carbodiimide and N‐hydroxysuccinimide (EDC/NHS). Because proteins typically contain many lysine groups, this strategy results in a heterogeneous distribution of tether positions. Genetically encoded peptide tags (e.g., ybbR) provide alternative chemistries for achieving site‐specific immobilization, but thus far a direct comparison of site‐specific vs. lysine‐based immobilization strategies to assess effects on the observed mechanical properties was lacking. Here, we compared lysine‐ vs. ybbR‐based protein immobilization in SMFS assays using several model polyprotein systems. Our results show that lysine‐based immobilization results in significant signal deterioration for monomeric streptavidin‐biotin interactions, and loss of the ability to correctly classify unfolding pathways in a multipathway Cohesin‐Dockerin system. We developed a mixed immobilization approach where a site‐specifically tethered ligand was used to probe surface‐bound proteins immobilized through lysine groups, and found partial recovery of specific signals. The mixed immobilization approach represents a viable alternative for mechanical assays on in vivo‐derived samples or other proteins of interest where genetically encoded tags are not feasible.
The surface immobilization technique for atomic force microscopy/single‐molecule force spectroscopy is crucial for experimental yield, accuracy and resolution. Reported herein is that a commonly used lysine‐based immobilization can result in significant signal deterioration, including lowering of rupture forces, effective shortening of linkers, and loss of the ability to correctly classify unfolding pathways in a multipathway polyprotein system.
Enzyme engineering is an important biotechnological process capable of generating tailored biocatalysts for applications in industrial chemical conversion and biopharma. Typical enhancements sought ...in enzyme engineering and
evolution campaigns include improved folding stability, catalytic activity, and/or substrate specificity. Despite significant progress in recent years in the areas of high-throughput screening and DNA sequencing, our ability to explore the vast space of functional enzyme sequences remains severely limited. Here, we review the currently available suite of modern methods for enzyme engineering, with a focus on novel readout systems based on enzyme cascades, and new approaches to reaction compartmentalization including single-cell hydrogel encapsulation techniques to achieve a genotype-phenotype link. We further summarize systematic scanning mutagenesis approaches and their merger with deep mutational scanning and massively parallel next-generation DNA sequencing technologies to generate mutability landscapes. Finally, we discuss the implementation of machine learning models for computational prediction of enzyme phenotypic fitness from sequence. This broad overview of current state-of-the-art approaches for enzyme engineering and evolution will aid newcomers and experienced researchers alike in identifying the important challenges that should be addressed to move the field forward.
Protein-conjugated magnetic nanoparticles (mNPs) are promising tools for a variety of biomedical applications, from immunoassays and biosensors to theranostics and drug-delivery. In such ...applications, conjugation of affinity proteins (e.g., antibodies) to the nanoparticle surface many times compromises biological activity and specificity, leading to increased reagent consumption and decreased assay performance. To address this problem, we engineered a biomolecular magnetic separation system that eliminates the need to chemically modify nanoparticles with the capture biomolecules or synthetic polymers of any kind. The system consists of (i) thermoresponsive magnetic iron oxide nanoparticles displaying poly(N-isopropylacrylamide) (pNIPAm), and (ii) an elastin-like polypeptide (ELP) fused with the affinity protein Cohesin (Coh). Proper design of pNIPAm-mNPs and ELP-Coh allowed for efficient cross-aggregation of the two distinct nanoparticle types under collapsing stimuli, which enabled magnetic separation of ELP-Coh aggregates bound to target Dockerin (Doc) molecules. Selective resolubilization of the ELP-Coh/Doc complexes was achieved under intermediate conditions under which only the pNIPAm-mNPs remained aggregated. We show that ELP-Coh is capable of magnetically separating and purifying nanomolar quantities of Doc as well as eukaryotic whole cells displaying the complementary Doc domain from diluted human plasma. This modular system provides magnetic enrichment and purification of captured molecular targets and eliminates the requirement of biofunctionalization of magnetic nanoparticles to achieve bioseparations. Our streamlined and simplified approach is amenable for point-of-use applications and brings the advantages of ELP-fusion proteins to the realm of magnetic particle separation systems.
We report the application of machine learning techniques to expedite classification and analysis of protein unfolding trajectories from force spectroscopy data. Using kernel methods, logistic ...regression, and triplet loss, we developed a workflow called Forced Unfolding and Supervised Iterative Online (FUSION) learning where a user classifies a small number of repeatable unfolding patterns encoded as images, and a machine is tasked with identifying similar images to classify the remaining data. We tested the workflow using two case studies on a multidomain XMod-Dockerin/Cohesin complex, validating the approach first using synthetic data generated with a Monte Carlo algorithm and then deploying the method on experimental atomic force spectroscopy data. FUSION efficiently separated traces that passed quality filters from unusable ones, classified curves with high accuracy, and identified unfolding pathways that were undetected by the user. This study demonstrates the potential of machine learning to accelerate data analysis and generate new insights in protein biophysics.
Directed evolution of oxidoreductases to improve their catalytic properties is being ardently pursued in the industrial, biotechnological, and biopharma sectors. Hampering this pursuit are current ...enzyme screening methods that are limited in terms of throughput, cost, time, and complexity. We present a directed evolution strategy that allows for large‐scale one‐pot screening of glucose oxidase (GOx) enzyme libraries in well‐mixed homogeneous solution. We used GOx variants displayed on the outer cell wall of yeasts to initiate a cascade reaction with horseradish peroxidase (HRP), resulting in peroxidase‐mediated phenol cross‐coupling and encapsulation of individual cells in well‐defined fluorescent alginate hydrogel shells within ~10 min in mixed cell suspensions. Following application of denaturing stress to whole‐cell GOx libraries, only cells displaying GOx variants with enhanced stability or catalytic activity were able to carry out the hydrogel encapsulation reaction. Fluorescence‐activated cell sorting was then used to isolate the enhanced variants. We characterized three of the newly evolved Aspergillus niger GOx enzyme sequences and found up to ~5‐fold higher specific activity, enhanced thermal stability, and differentiable glycosylation patterns. By coupling intracellular gene expression with the rapid formation of an extracellular hydrogel capsule, our system improves high‐throughput screening for directed evolution of H
2O
2‐producing enzymes many folds.
Directed evolution of oxidoreductases to improve their catalytic properties is being ardently pursued in the industrial, biotechnological, and biopharma sectors. Hampering this pursuit are current enzyme screening methods that are limited in terms of throughput, cost, time and complexity. We present a hydrogel‐based directed evolution strategy that allows for large‐scale one‐pot screening of glucose oxidase (GOx) enzyme libraries in well‐mixed homogeneous solution.
We investigated the influence of fluorination on unfolding and unbinding reaction pathways of a mechanostable protein complex comprising the tandem dyad XModule-Dockerin bound to Cohesin. Using ...single-molecule atomic force spectroscopy, we mapped the energy landscapes governing the unfolding and unbinding reactions. We then used sense codon suppression to substitute trifluoroleucine in place of canonical leucine globally in XMod-Doc. Although TFL substitution thermally destabilized XMod-Doc, it had little effect on XMod-Doc:Coh binding affinity at equilibrium. When we mechanically dissociated global TFL-substituted XMod-Doc from Coh, we observed the emergence of a new unbinding pathway with a lower energy barrier. Counterintuitively, when fluorination was restricted to Doc, we observed mechano-stabilization of the non-fluorinated neighboring XMod domain. This suggests that intramolecular deformation is modulated by fluorination and highlights the differences between equilibrium thermostability and non-equilibrium mechanostability. Future work is poised to investigate fluorination as a means to modulate mechanical properties of synthetic proteins and hydrogels.
Bacterial colonization of the human intestine requires firm adhesion of bacteria to insoluble substrates under hydrodynamic flow. Here we report the molecular mechanism behind an ultrastable protein ...complex responsible for resisting shear forces and adhering bacteria to cellulose fibers in the human gut. Using single-molecule force spectroscopy (SMFS), single-molecule FRET (smFRET), and molecular dynamics (MD) simulations, we resolve two binding modes and three unbinding reaction pathways of a mechanically ultrastable R. champanellensis (Rc) Dockerin:Cohesin (Doc:Coh) complex. The complex assembles in two discrete binding modes with significantly different mechanical properties, with one breaking at ~500 pN and the other at ~200 pN at loading rates from 1-100 nN s
. A neighboring X-module domain allosterically regulates the binding interaction and inhibits one of the low-force pathways at high loading rates, giving rise to a catch bonding mechanism that manifests under force ramp protocols. Multi-state Monte Carlo simulations show strong agreement with experimental results, validating the proposed kinetic scheme. These results explain mechanistically how gut microbes regulate cell adhesion strength at high shear stress through intricate molecular mechanisms including dual-binding modes, mechanical allostery and catch bonds.
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