ArticleJournal of the American Chemical Society2017
Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation.
Article in Journal of the American Chemical Society, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 37 papers.
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Who cites it
37 citing papers in PubMed, 73 citations in OpenAlex.
- Advancements and applications of click chemistry in protein labeling and bioconjugation.RSC advances · 2026Review
- Evolution of N-terminal mechanical lability as a determinant for Type III secretion.Protein science : a publication of the Protein Society · 2026Article
- Silanes to Silatranes: Robust Functionalization for Single-Molecule Force Spectroscopy.Chembiochem : a European journal of chemical biology · 2026Article
- Single Molecule Force Spectroscopy to Probe Intermediates and Energetics of Membrane Protein Folding.Chemical reviews · 2026Review
- A Type III secretion system effector evolved to be mechanically labile and initiate unfolding from the N-terminus.bioRxiv : the preprint server for biology · 2026Article
- Highly Branched Sulfated Glycopolymers as Mucin Mimetics.Journal of the American Chemical Society · 2025Article
- Structural Basis of High-Precision Protein Ligation and Its Application.Journal of the American Chemical Society · 2025Article
- Direct observation of prion-like propagation of protein misfolding templated by pathogenic mutants.Nature chemical biology · 2024Article
- Mechanistic Insight into the Mechanical Unfolding of the Integral Membrane Diacylglycerol Kinase.JACS Au · 2024Article
- Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
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- Energy landscapes of fast-folding proteins pushing the limits of atomic force microscope (AFM) pulling.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc.International journal of molecular sciences · 2020Article
- Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs.Chembiochem : a European journal of chemical biology · 2020Article
- Piecewise All-Atom SMD Simulations Reveal Key Secondary Structures in Luciferase Unfolding Pathway.Biophysical journal · 2020Article
- Enzymatic Construction of Protein Polymer/Polyprotein Using OaAEP1 and TEV Protease.Bio-protocol · 2020Article
- Review
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Authors and funding
11 authors at 2 institutions in 1 country.
Funding
Abstract
Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize mechanical properties of biomolecules. Historically, accessibility relies upon the nonspecific adhesion of biomolecules to a surface and a cantilever and, for proteins, the integration of the target protein into a polyprotein. However, this assay results in a low yield of high-quality data, defined as the complete unfolding of the polyprotein. Additionally, nonspecific surface adhesion hinders studies of α-helical proteins, which unfold at low forces and low extensions. Here, we overcame these limitations by merging two developments: (i) a polyprotein with versatile, genetically encoded short peptide tags functionalized via a mechanically robust Hydrazino-Pictet-Spengler ligation and (ii) the efficient site-specific conjugation of biomolecules to PEG-coated surfaces. Heterobifunctional anchoring of this polyprotein construct and DNA via copper-free click chemistry to PEG-coated substrates and a strong but reversible streptavidin-biotin linkage to PEG-coated AFM tips enhanced data quality and throughput. For example, we achieved a 75-fold increase in the yield of high-quality data and repeatedly probed the same individual polyprotein to deduce its dynamic force spectrum in just 2 h. The broader utility of this polyprotein was demonstrated by measuring three diverse target proteins: an α-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (α
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.