A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues

Rivas‐Pardo, Jaime Andrés; Li, Yong; Mártonfalvi, Zsolt; Tapia‐Rojo, Rafael; Unger, Andreas; Fernández-Trasancos, Ángel; Herrero-Galán, Elías; Velázquez-Carreras, Diana; Fernández, Julio M.; Linke, Wolfgang A.; Alegre-Cebollada, Jorge

doi:10.1038/s41467-020-15465-9

Cited by 47 publications

(55 citation statements)

References 82 publications

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“…4A, middle). Thereafter, the kinetics were similar to Wt fibers, indicating that most cleavable titins were severed after ~10 min of treatment, slightly faster than previously reported by us (Rivas-Pardo et al, 2020), due probably to different activities of the TEV-protease batches used. Protein gels confirmed ~50% cleavage for Het TC samples and ~100% cleavage for Hom (Fig.…”

Section: Resultssupporting

confidence: 74%

“…TC mice (Rivas-Pardo et al, 2020) were bred and housed at the University Clinic Muenster. Approval from the local authorities was obtained (LANUV NRW, 81-02.04.2019.A472).…”

Section: Methodsmentioning

confidence: 99%

“…To mitigate the problems outlined above, a new mouse model was generated with a cloned-in HaloTag-TEV cassette inserted into I-band titin close to the A-band (Rivas-Pardo et al, 2020). We refer to this mouse as the titin cleavage (TC) model.…”

Section: Introductionmentioning

confidence: 99%

“…for the assessment of titin cleaving. The insertion itself does not affect mouse development, muscle structure, or performance (Rivas-Pardo et al, 2020). Our experimental strategy is to use TEV protease to cleave I-band titin and terminate its functionality in a targeted and controllable fashion.…”

Section: Introductionmentioning

confidence: 99%

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Graded titin cleavage progressively reduces tension and uncovers the source of A-band stability in contracting muscle

Li¹,

Hessel²,

Unger³

et al. 2020

Preprint

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The giant muscle protein titin is a major contributor to passive force; however, its role in active force generation is unresolved. Here, we use a novel titin-cleavage (TC) mouse model that allows specific and rapid cutting of elastic titin to quantify how titin-based forces define myocyte ultrastructure and mechanics. We show that under mechanical strain, as titin cleavage doubles from heterozygous to homozygous TC muscles, Z-disks become increasingly out of register while passive and active forces are reduced. Interactions of elastic titin with sarcomeric actin filaments are revealed. Strikingly, when titin-cleaved muscles contract, myosin-containing A-bands become split and adjacent myosin filaments move in opposite directions while also shedding myosins. This establishes intact titin filaments as critical force-transmission networks, buffering the forces observed by myosin filaments during contraction. To perform this function, elastic titin must change stiffness or extensible length, unveiling its fundamental role as an activation-dependent spring in contracting muscle.

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Section: Resultssupporting

confidence: 74%

“…TC mice (Rivas-Pardo et al, 2020) were bred and housed at the University Clinic Muenster. Approval from the local authorities was obtained (LANUV NRW, 81-02.04.2019.A472).…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Graded titin cleavage progressively reduces tension and uncovers the source of A-band stability in contracting muscle

Li¹,

Hessel²,

Unger³

et al. 2020

Preprint

Self Cite

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“…Mechanical forces are fundamental in biology, and protein mechanical stability is an important non-equilibrium parameter that can influence behavior in vivo. 26,27 Mechanical stability describes how much tension a folded domain can withstand prior to unfolding, or how much force is required to dissociate a receptor-ligand complex. Singlemolecule force spectroscopy (SMFS) [28][29][30][31] with the atomic force microscope (AFM) can be used to stretch single protein molecules, quantify intermediate folding states 32,33 elucidate [un]folding energy landscapes while accounting for differences in loading geometry [34][35][36][37][38] or the presence of dual modes of ligand recognition 39,40 .…”

Section: Main Text Introductionmentioning

confidence: 99%

Influence of Fluorination on Single-Molecule Unfolding and Rupture Pathways of a Mechanostable Protein Adhesion Complex

Yang

Liu

et al. 2020

Preprint

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AbstractFluorination of proteins by cotranslational incorporation of non-canonical amino acids is a valuable tool for enhancing biophysical stability. Despite many prior studies investigating the effects of fluorination on equilibrium stability, its influence on non-equilibrium mechanical stability remains unknown. Here, we used single-molecule force spectroscopy (SMFS) with the atomic force microscope (AFM) to investigate the influence of fluorination on unfolding and unbinding pathways of a mechanically ultrastable bacterial adhesion complex. We assembled modular polyproteins comprising the tandem dyad XModule-Dockerin (XMod-Doc) bound to a globular Cohesin (Coh) domain. By applying tension across the binding interface, and quantifying single-molecule unfolding and rupture events, we mapped the energy landscapes governing the unfolding and unbinding reactions. We then used sense codon suppression to substitute trifluoroleucine (TFL) in place of canonical leucine (LEU) globally in XMod-Doc, or selectively within the Doc subdomain of a mutant 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. These results suggest that intramolecular deformation networks can be modulated by fluorination, and further highlight significant differences between equilibrium thermostability, where all constructs were destabilized, and non-equilibrium mechanostability, where XMod was strengthened. Future work is poised to investigate the influence of non-natural amino acids on mechanically-accelerated protein unfolding and unbinding reaction pathways.

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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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A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues

Cited by 47 publications

References 82 publications

Graded titin cleavage progressively reduces tension and uncovers the source of A-band stability in contracting muscle

Graded titin cleavage progressively reduces tension and uncovers the source of A-band stability in contracting muscle

Influence of Fluorination on Single-Molecule Unfolding and Rupture Pathways of a Mechanostable Protein Adhesion Complex

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

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