Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy
| Autor: | Michael A. Nash, Hermann E. Gaub, Markus A. Jobst, Magnus S. Bauer, Lukas F. Milles, Wolfgang Ott, Ellis Durner |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Materials science Protein Conformation General Physics and Astronomy Nanotechnology 02 engineering and technology Polyethylene Glycols 03 medical and health sciences chemistry.chemical_compound Mechanobiology PEG ratio Escherichia coli Molecule General Materials Science Amino Acids Protein Unfolding chemistry.chemical_classification Bioconjugation Biomolecule General Engineering Force spectroscopy Polymer 021001 nanoscience & nanotechnology Elasticity Single Molecule Imaging Biomechanical Phenomena Elastin 030104 developmental biology Immobilized Proteins chemistry Biophysics 0210 nano-technology Peptides Ethylene glycol |
| Zdroj: | ACS Nano |
| ISSN: | 1936-086X |
| Popis: | Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality. In many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used to anchor molecules of interest to surfaces and/or cantilever tips. The limitation, however, is that PEG exhibits a well-known trans-trans-gauche to all-trans transition, which results in marked deviation from standard polymer elasticity models such as the worm-like chain, particularly at elevated forces. As a result, the assignment of unfolding events to protein domains based on their corresponding amino acid chain lengths is significantly obscured. Here, we provide a solution to this problem by implementing unstructured elastin-like polypeptides as linkers to replace PEG. We investigate the suitability of tailored elastin-like polypeptides linkers and perform direct comparisons to PEG, focusing on attributes that are critical for single-molecule force experiments such as linker length, monodispersity, and bioorthogonal conjugation tags. Our results demonstrate that by avoiding the ambiguous elastic response of mixed PEG/peptide systems and instead building the molecular mechanical systems with only a single bond type with uniform elastic properties, we improve data quality and facilitate data analysis and interpretation in force spectroscopy experiments. The use of all-peptide linkers allows alternative approaches for precisely defining elastic properties of proteins linked to surfaces. |
| Databáze: | OpenAIRE |
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