期刊
NANO LETTERS
卷 15, 期 10, 页码 7091-7098出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.nanolett.5b03166
关键词
AFM; atomic force microscopy; protein folding; single-molecule force spectroscopy; focused-ion-beam milling; cantilever dynamics; single-molecule biophysics
类别
资金
- National Research Council
- NIH biophysics training grant [T32 GM065103]
- National Science Foundation [DBI-135398, Phys-1125844]
- NIH [R01 AI080709]
- Butcher Grant
- NIST
- Division Of Physics
- Direct For Mathematical & Physical Scien [1125844] Funding Source: National Science Foundation
- Div Of Biological Infrastructure
- Direct For Biological Sciences [1353987] Funding Source: National Science Foundation
Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard commercial cantilever is 50-1000 mu s, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-mu s temporal resolution was achieved using an ultrashort (L = 9 mu m) cantilever on a custom-built, high-speed AFM. By micro-machining such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected the modified cantilevers on a commercial AFM retrofitted with a detection laser system featuring a 3-mu m circular spot size. Moreover, individual cantilevers were reused over multiple days. The improved capabilities of the modified cantilevers for SMFS were showcased by unfolding a polyprotein, a popular biophysical assay. Specifically, these cantilevers maintained a 1-mu s response time while eliminating cantilever ringing (Q congruent to 0.5). We therefore expect such cantilevers, along with the instrumentational improvements to detect them on a commercial AFM, to accelerate high-precision AFM-based SMFS studies.
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