Quasi-simultaneous imaging/pulling analysis of single polyprotein molecules by atomic force microscopy
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(Color) Diagram of our AFM apparatus. This setup is based on a custom-built AFM puller previously described in which the data acquisition boards used for the pulling experiments are commanded by custom-made IGOR PRO software (Ref. 9). The AFM imaging section was added by integrating a high voltage unit (Dulcinea; Nanotec Electrónica S.L., Madrid, Spain) combined with a digital signal processor board (M6701 floating point DSP board, Innovative Integration, Simi Valley, CA, USA) commanded by WSXM proprietary software. Displacements are measured using the capacitive sensors, and the resulting signals are fed to the pulling and imaging controllers. Both controllers have been communicated by means of a custom-made software module (available upon request). A dithering piezo for dynamic operation was installed below the fluid cell. Further details on the components, connectivity and synchronization of both AFM sections are described on Sec. II).
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(Color) Control AFM experiments using I27 polyproteins. (A) Typical recording using standard SMSF: in this force vs tip-surface distance curve each peak represents the unfolding of a single module in a polyprotein molecule (in this recording seven modules were stretched; last peak marks the end of the experiment and typically corresponds to the detachment of the polyprotein molecule). The orange lines represent fittings to the wormlike chain model of polymer elasticity (deviation observed in the first modules is attributed to the presence of an unfolding intermediate in the I27 module) (Ref. 4). (B) Topographical characterization of the substrate (a gold-coated coverslip under PBS buffer). (C) This substrate after incubation with the polyprotein sample. The insets are profiles taken at the indicated lines (red on B; green on C). The black circle on (C) indicates an attempt of stretching a putative polyprotein structure by SMSF.
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(Color) Quasi-simultaneous imaging/force spectroscopy of I27 polyproteins. (a) trials on single molecules. Here we show the equalized derivative topography of a clean flattened gold-coated coverslip (left) and the same substrate with polyprotein at incubated over night (right). We focused our analysis on putative single molecules (black circle in the right image and left profile, height of , indentation of ), but we were not able to obtain any sawtooth pattern (right trace). (b) AFM imaging/SMFS at high protein concentration . From a straight topograph we selected a particle with bigger dimensions than those expected for single molecules (green arrow, profile showing height of ). We could do several substrate-to-tip approach/retraction cycles (right) and obtained several sawtooth patterns, which after fitting the peaks to a family of wormlike chain curves (not shown), yielded similar parameters to those previously reported for I27 modules (Ref. 6). Straight topographs are color coded so that in darker is deeper and lighter is higher. Each polyprotein molecule (with 8 or 12 globular monomers) in solution is expected, from polymer physics, to be coiled. The overall dimensions of each monomer, measured from its atomic structure, are . In the case of the 12-mer the expected (extended) length of each polyprotein measured by rotary shadowing EM is (Ref. 22).
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