Single-Molecule Adhesion of a Stimuli-Responsive Oligo(ethylene glycol) Copolymer to Gold

Adhesion of environmentally responsive polymers to biocompatible surfaces is an important issue that has been explored in several nanobiotechnology applications. Here, we prepared multi-responsive statistical copolymers of two oligo(ethylene glycol) methyl ether methacrylate macromonomers with differing ethylene glycol side chain lengths using RAFT polymerization. The lower critical solution temperature of the copolymers was characterized using visible light extinction, and the chemical composition and molecular weight were measured using NMR spectroscopy and size-exclusion chromatography, respectively. The characterization results demonstrated that the transition temperature could be controlled by varying the macromonomer feed ratios, and the molecular weight could be controlled by varying the amount of the RAFT chain transfer agent in the polymerization feed. Using AFM single-molecule force spectroscopy, we measured the adhesion characteristics of single copolymer molecules to a gold surface. We found that dehydration and collapse of the copolymer in a high ionic strength buffer resulted in dramatically reduced bridging length distributions that maintained their single-molecule bimodal character. In the collapsed state, the polymer exhibited a lower absolute desorption force while cooperativity effects were found to increase the desorption force per chain for multi-chain interactions. Our results confirmed that the polymer in a collapsed conformation exhibited a dramatically reduced volume occupancy above the gold surface. These results demonstrate at the single-molecule level how solvent-induced collapse of an environmentally responsive copolymer modulates surface adhesion forces and bridging length distributions in a controllable way

Iterative Machine Learning for Classification and Discovery of Single-Molecule Unfolding Trajectories from Force Spectroscopy Data

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

Mixed Stimuli-Responsive Magnetic and Gold Nanoparticle System for Rapid Purification, Enrichment, and Detection of Biomarkers

A new diagnostic system for the enrichment and detection of protein biomarkers from human plasma is presented. Gold nanoparticles (AuNPs) were surface-modified with a diblock copolymer synthesized using reversible addition−fragmentation chain transfer (RAFT) polymerization. The diblock copolymer contained a thermally responsive poly(N-isopropylacrylamide) (pNIPAAm) block, a cationic amine-containing block, and a semi-telechelic PEG2-biotin end group. When a mixed suspension of 23 nm pNIPAAm-modified AuNPs was heated with pNIPAAm-coated 10 nm iron oxide magnetic nanoparticles (mNPs) in human plasma, the thermally responsive pNIPAAm directed the formation of mixed AuNP/mNP aggregates that could be separated efficiently with a magnet. Model studies showed that this mixed nanoparticle system could efficiently purify and strongly enrich the model biomarker protein streptavidin in spiked human plasma. A 10 ng/mL streptavidin sample was mixed with the biotinylated pNIPAAm-modified AuNPs and magnetically separated in the mixed nanoparticle system with pNIPAAm mNPs. The aggregates were concentrated into a 50-fold smaller fluid volume at room temperature where the gold nanoparticle reagent redissolved with the streptavidin target still bound. The concentrated gold-labeled streptavidin could be subsequently analyzed directly using lateral flow immunochromatography. This rapid capture and enrichment module thus utilizes the mixed stimuli-responsive nanoparticle system to achieve concentration of a gold-labeled biomarker that can be directly analyzed using lateral flow or other rapid diagnostic strategies

Optimal Sacrificial Domains in Mechanical Polyproteins: S. epidermidis Adhesins Are Tuned for Work Dissipation

The opportunistic pathogen Staphylococcus epidermidis utilizes a multidomain surface adhesin protein to bind host components and adhere to tissues. While it is known that the interaction between the SdrG receptor and its fibrinopeptide target (FgB) is exceptionally mechanostable (∼2 nN), the influence of downstream B domains (B1 and B2) is unclear. Here, we studied the mechanical relationships between folded B domains and the SdrG receptor bound to FgB. We used protein engineering, single-molecule force spectroscopy (SMFS) with an atomic force microscope (AFM), and Monte Carlo simulations to understand how the mechanical properties of folded sacrificial domains, in general, can be optimally tuned to match the stability of a receptor–ligand complex. Analogous to macroscopic suspension systems, sacrificial shock absorber domains should neither be too weak nor too strong to optimally dissipate mechanical energy. We built artificial molecular shock absorber systems based on the nanobody (VHH) scaffold and studied the competition between domain unfolding and receptor unbinding. We quantitatively determined the optimal stability of shock absorbers that maximizes work dissipation on average for a given receptor and found that natural sacrificial domains from pathogenic S. epidermidis and Clostridium perfringens adhesins exhibit stabilities at or near this optimum within a specific range of loading rates. These findings demonstrate how tuning the stability of sacrificial domains in adhesive polyproteins can be used to maximize mechanical work dissipation and serve as an adhesion strategy by bacteria

Multiplexed Enrichment and Detection of Malarial Biomarkers Using a Stimuli-Responsive Iron Oxide and Gold Nanoparticle Reagent System

There is a need for simple yet robust biomarker and antigen purification and enrichment strategies that are compatible with current rapid diagnostic modalities. Here, a stimuli-responsive nanoparticle system is presented for multiplexed magneto-enrichment and non-instrumented lateral flow strip detection of model antigens from spiked pooled plasma. The integrated reagent system allows purification and enrichment of the gold-labeled biomarker half-sandwich that can be applied directly to lateral flow test strips. A linear diblock copolymer with a thermally responsive poly(<i>N</i>-isopropylacrylamide) (pNIPAm) segment and a gold-binding block composed of NIPAm-<i>co</i>-<i>N</i>,<i>N</i>-dimethylaminoethylacrylamide was prepared by reversible addition–fragmentation chain transfer polymerization. The diblock copolymer was used to functionalize gold nanoparticles (AuNPs), with subsequent bioconjugation to yield thermally responsive pNIPAm-AuNPs that were co-decorated with streptavidin. These AuNPs efficiently complexed biotinylated capture antibody reagents that were bound to picomolar quantities of pan-aldolase and Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in spiked pooled plasma samples. The gold-labeled biomarker half-sandwich was then purified and enriched using 10 nm thermally responsive magnetic nanoparticles that were similarly decorated with pNIPAm. When a thermal stimulus was applied in conjunction with a magnetic field, coaggregation of the AuNP half-sandwiches with the pNIPAm-coated iron oxide nanoparticles created large aggregates that were efficiently magnetophoresed and separated from bulk serum. The purified biomarkers from a spiked pooled plasma sample could be concentrated 50-fold into a small volume and applied directly to a commercial multiplexed lateral flow strip to dramatically improve the signal-to-noise ratio and test sensitivity

“Smart” Diblock Copolymers as Templates for Magnetic-Core Gold-Shell Nanoparticle Synthesis

We report a new strategy for synthesizing temperature-responsive γ-Fe2O3-core/Au-shell nanoparticles (Au-mNPs) from diblock copolymer micelles. The amphiphilic diblock copolymer chains were synthesized using reversible addition−fragmentation chain-transfer (RAFT) with a thermally responsive “smart” poly(N-isopropylacrylamide) (pNIPAAm) block and an amine-containing poly(N,N-dimethylaminoethylacrylamide) (DMAEAm) block that acted as a reducing agent during gold shell formation. The Au-mNPs reversibly aggregated upon heating the solution above the transition temperature of pNIPAAm, resulting in a red-shifted localized surface plasmon resonance

Titrating Avidity of Yeast-Displayed Proteins Using a Transcriptional Regulator

Yeast surface display is a valuable tool for protein engineering and directed evolution; however, significant variability in the copy number (i.e., avidity) of displayed variants on the yeast cell wall complicates screening and selection campaigns. Here, we report an engineered titratable display platform that modulates the avidity of Aga2-fusion proteins on the yeast cell wall dependent on the concentration of the anhydrotetracycline (aTc) inducer. Our design is based on a genomic Aga1 gene copy and an episomal Aga2-fusion construct both under the control of an aTc-dependent transcriptional regulator that enables stoichiometric and titratable expression, secretion, and display of Aga2-fusion proteins. We demonstrate tunable display levels over 2–3 orders of magnitude for various model proteins, including glucose oxidase enzyme variants, mechanostable dockerin-binding domains, and anti-PDL1 affibody domains. By regulating the copy number of displayed proteins, we demonstrate the effects of titratable avidity levels on several specific phenotypic activities, including enzyme activity and cell adhesion to surfaces under shear flow. Finally, we show that titrating down the display level allows yeast-based binding affinity measurements to be performed in a regime that avoids ligand depletion effects while maintaining small sample volumes, avoiding a well-known artifact in yeast-based binding assays. The ability to titrate the multivalency of proteins on the yeast cell wall through simple inducer control will benefit protein engineering and directed evolution methodology relying on yeast display for broad classes of therapeutic and diagnostic proteins of interest

Mapping Mechanostable Pulling Geometries of a Therapeutic Anticalin/CTLA‑4 Protein Complex

We used single-molecule AFM force spectroscopy (AFM-SMFS) in combination with click chemistry to mechanically dissociate anticalin, a non-antibody protein binding scaffold, from its target (CTLA-4), by pulling from eight different anchor residues. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in koff by 2–3 orders of magnitude over a force range of 50–200 pN. Five of the six internal anchor points gave rise to complexes significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1–10 nN s–1. Anisotropic network modeling and molecular dynamics simulations helped to explain the geometric dependency of mechanostability. These results demonstrate that optimization of attachment residue position on therapeutic binding scaffolds can provide large improvements in binding strength, allowing for mechanical affinity maturation under shear stress without mutation of binding interface residues

Mechanisms of Nanonewton Mechanostability in a Protein Complex Revealed by Molecular Dynamics Simulations and Single-Molecule Force Spectroscopy

Can molecular dynamics simulations predict the mechanical behavior of protein complexes? Can simulations decipher the role of protein domains of unknown function in large macromolecular complexes? Here, we employ a wide-sampling computational approach to demonstrate that molecular dynamics simulations, when carefully performed and combined with single-molecule atomic force spectroscopy experiments, can predict and explain the behavior of highly mechanostable protein complexes. As a test case, we studied a previously unreported homologue from Ruminococcus flavefaciens called X-module-Dockerin (XDoc) bound to its partner Cohesin (Coh). By performing dozens of short simulation replicas near the rupture event, and analyzing dynamic network fluctuations, we were able to generate large simulation statistics and directly compare them with experiments to uncover the mechanisms involved in mechanical stabilization. Our single-molecule force spectroscopy experiments show that the XDoc-Coh homologue complex withstands forces up to 1 nN at loading rates of 105 pN/s. Our simulation results reveal that this remarkable mechanical stability is achieved by a protein architecture that directs molecular deformation along paths that run perpendicular to the pulling axis. The X-module was found to play a crucial role in shielding the adjacent protein complex from mechanical rupture. These mechanisms of protein mechanical stabilization have potential applications in biotechnology for the development of systems exhibiting shear enhanced adhesion or tunable mechanics