期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 139, 期 29, 页码 9867-9875出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacs.7b02958
关键词
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资金
- National Institutes of Health (NIH) Molecular Biophysics Training Grant [T32 GM-065103]
- National Research Council
- National Science Foundation [DBI-1353987, Phys-1125844]
- NIH [R01 AI080709, S10 RR026641]
- Butcher Grant
- NIST
- Direct For Biological Sciences
- Div Of Biological Infrastructure [1353987] Funding Source: National Science Foundation
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 alpha-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 alpha-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (alpha D-3). Indeed, at low loading rates, a3D represents the most mechanically labile protein yet characterized by AFM. Such efficient SMFS studies on a commercial AFM enable the rapid characterization of macromolecular folding over a broader range of proteins and a wider array of experimental conditions (pH, temperature, denaturants). Further, by integrating these enhancements with optical traps, we demonstrate how efficient bioconjugation to otherwise nonstick surfaces can benefit diverse single-molecule studies.
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