Correlating single-molecule rupture mechanics with cell population adhesion by yeast display

Santos, Mariana Sá; Liu, Haipei; Schittny, Valentin; Vanella, Rosario; Nash, Michael A.

doi:10.1016/j.bpr.2021.100035

Cited by 8 publications

(14 citation statements)

References 46 publications

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“…In the SDA, cells are adhered onto a ligand-functionalized coverglass and undergo a spinning procedure that generates a gradient of hydrodynamic shear stress to remove cells. The shear stress at which 50% of the cells are observed to detach (τ 50 ) can be used to quantify the average cell adhesion strength for the cell population. ,,, …”

Section: Resultsmentioning

confidence: 99%

“…The shear stress at which 50% of the cells are observed to detach (τ 50 ) can be used to quantify the average cell adhesion strength for the cell population. 60,61,68,69 In our implementation of the SDA, yeast adhesion is mediated primarily by the displayed POI and should therefore be strongly influenced by protein levels at the cell surface. The display level was titrated with different aTc concentrations for 4 h of induction, and analytic flow cytometry was performed to verify successful titration of the fraction of cells displaying XDocIII and the amount of displayed protein per cell.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…The shear stress at which 50% of the cells are observed to detach (τ 50 ) can be used to quantify the average cell adhesion strength for the cell population. 60 , 61 , 68 , 69 …”

Section: Resultsmentioning

confidence: 99%

“…An in-house device based on a spinning disk cell adhesion assay was employed to study cell population adhesion profiles. The construction of the apparatus and coverglasses’ preparation were performed as described previously. , Briefly, aminosilanized coverglasses were functionalized with Cohesin E (CohE–HIS–ybbR) using a 250 μM solution of Maleimide–PEG–NHS (5 kDa; Rapp Polymere, Tübingen, Germany) in 25 mM CH 3 O–PEG–NHS (5 kDa; Rapp Polymere, Tübingen, Germany) and 100 mM HEPES pH 8 for 30 min. Coverglasses were rinsed with ultrapure water and incubated with 200 μM Coenzyme A (CoA) solution for 30 min.…”

Section: Methodsmentioning

confidence: 99%

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Titrating Avidity of Yeast-Displayed Proteins Using a Transcriptional Regulator

et al. 2023

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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.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

“…The shear stress at which 50% of the cells are observed to detach (τ 50 ) can be used to quantify the average cell adhesion strength for the cell population. 60 , 61 , 68 , 69 …”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Titrating Avidity of Yeast-Displayed Proteins Using a Transcriptional Regulator

et al. 2023

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“…We first conducted AFM-SMFS on complexes consisting of a monomeric variant of streptavidin (mSA) [41][42][43] bound to biotin, where mSA was immobilized on a glass surface and PEG-biotin was attached to a cantilever. As one of the first receptor-ligand systems measured in AFM-SMFS [44] , the streptavidin-biotin interaction has been well characterized while the newly developed monomeric variants with unambiguous binding stoichiometry shows slightly different rupture forces compared to those in tetramer form [45,46] .…”

Section: Lysine Vs Site-specific Linkage For Monomeric Streptavidin-b...mentioning

confidence: 99%

Direct Comparison of Lysine vs. Site-specific Protein Surface Immobilization in Single-molecule Mechanical Assays

Liu

Santos

et al. 2023

Preprint

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Single-molecule force spectroscopy (SMFS) is a powerful method for studying folding states and mechanical properties of proteins, however, it requires surface immobilization of proteins onto force-transducing probes such as cantilevers or microscale beads. A common immobilization method relies on coupling surface-exposed 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 in the molecule. 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 directly 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.

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Direct Comparison of Lysine versus Site‐Specific Protein Surface Immobilization in Single‐Molecule Mechanical Assays**

Liu

Santos

et al. 2023

Angewandte Chemie

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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-(3dimethyl-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 vivoderived samples or other proteins of interest where genetically encoded tags are not feasible.

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Correlating single-molecule rupture mechanics with cell population adhesion by yeast display

Cited by 8 publications

References 46 publications

Titrating Avidity of Yeast-Displayed Proteins Using a Transcriptional Regulator

Titrating Avidity of Yeast-Displayed Proteins Using a Transcriptional Regulator

Direct Comparison of Lysine vs. Site-specific Protein Surface Immobilization in Single-molecule Mechanical Assays

Direct Comparison of Lysine versus Site‐Specific Protein Surface Immobilization in Single‐Molecule Mechanical Assays**

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