Kinetics of Surface-Driven Self-Assembly and Fatigue-Induced Disassembly of a Virus- Based Nanocoating  Alejandro Valbuena, Mauricio G. Mateu  Biophysical Journal  Volume 112, Issue 4, Pages 663-673 (February 2017) DOI: 10.1016/j.bpj.2016.11.3209 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 The HIV CA lattice and self-assembly onto a solid substrate. (a) Left: ribbon model of the CA protein monomer. The NTD (red) and CTD (blue) are joined by a flexible linker (green); center: six CA monomers joined in a hexamer through NTD-NTD and NTD-CTD interactions; right: CA hexamers in the CA lattices are joined through CTD-CTD interactions. (b) High-resolution AFM image of a part of the 2D CA lattice self-assembled on a mica surface; a calibrated height scale is shown at left; the full scale spans 1 nm. The horizontal scale bar represents 15 nm. (c) Scheme of the experimental setup. A 71 mm2 mica disc (side view; black) on a AFM stage is fully covered by a drop of a buffered aqueous solution containing (at the concentrations tested in this study) CA monomers (red dots) in equilibrium with a few CA dimers. CA self-assembles into a hexagonal lattice by growing into patches (red extended areas) that eventually merge with each other. Biophysical Journal 2017 112, 663-673DOI: (10.1016/j.bpj.2016.11.3209) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Kinetics of self-assembly of the CA lattice on a solid substrate, followed by AFM. (a) Freshly purified CA. Upper row, left to right: images taken at 1 μM CA (total monomer concentration) at incubation times of 6 s, 1 min, 5 min, and 30 min. Middle row, left to right: images taken at 0.6 μM CA at 10 min, 1 h, and 4 h. Bottom row: images taken at 0.3 μM CA at 2 and 4 h. (b) The same CA stock after aging at 4°C for 10 month. Upper row, left to right: images taken at 1 μM CA at incubation times of 1, 3, 20, and 60 min. Middle row, left to right: images taken at 0.6 μM CA at 10, 20, and 60 min. Bottom row: images taken at 0.45 μM CA at 1 and 3 h. A calibrated height scale is shown at left; the full scale spans 10 nm. The horizontal scale bars represent 400 nm. To see this figure in color, go online. Biophysical Journal 2017 112, 663-673DOI: (10.1016/j.bpj.2016.11.3209) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Kinetics of self-assembly of the CA lattice on a solid substrate. Substrate surface area covered as a function of time during self-assembly of the CA lattice. Experimental data points (symbols) were fitted to sigmoidal Hill functions (Eq. 1) (continuous lines). (a) Freshly purified CA at the following (monomer) concentrations 1 μM (squares), 0.6 μM (triangles), and 0.3 μM (diamonds). (b) The same CA stock after aging at 4°C for 10 month, at the following concentrations: 1 μM (squares), 0.72 μM (circles), 0.6 μM (triangles), and 0.46 μM (diamonds). In (a) and (b), the data obtained at 1 μM CA are joined by a continuous line for a better appreciation of the deviation relative to the sigmoidal fitting curve at the highest tested concentration. (c) Value of the cooperative parameter n as a function of [CA]. (d) Value of t50 as a function of [CA]. In (c) and (d), triangles and squares respectively correspond to a fresh (a) or aged (b) protein sample. Error bars in (a) and (b) correspond to SDs of experimental values. Bars in (c) and (d) correspond to fitting errors derived from the fittings in (a) and (b). To see this figure in color, go online. Biophysical Journal 2017 112, 663-673DOI: (10.1016/j.bpj.2016.11.3209) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 Kinetics of disassembly of the CA lattice on a solid substrate in the absence of applied force, followed in real time by AFM. (a) A CA sheet was assembled to fully coat a mica disk. Immediately after coating was completed, free CA was removed by washing with PBS, and the lattice submerged in PBS was left to spontaneously dissociate. Images shown correspond to 0, 6, 8, and 12 h of disassembly time. A calibrated height scale is shown at left; the full scale spans 10 nm. The horizontal scale bars represent 400 nm. (b) Percent surface area that remains covered as a function of time during disassembly of the CA lattice in the absence of applied force. Data were fitted to a sigmoidal curve (continuous line). Error bars correspond to SD of experimental values. To see this figure in color, go online. Biophysical Journal 2017 112, 663-673DOI: (10.1016/j.bpj.2016.11.3209) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 Disassembly of the CA lattice on a solid substrate under applied force, followed in real time by AFM imaging. A CA sheet was assembled to fully coat a mica disk. Immediately after coating was completed, free CA was removed by washing with PBS. A selected area of a CA lattice was chosen and intermittently subjected to mechanical force; the effect of different forces (a) or indentation frequency (b) (values indicated at left) were tested to induce materials fatigue within the indented area. AFM images were taken at different times, and time-lapse Movies S1–S6 were obtained. A calibrated height scale is shown at left; the full scale spans 10 nm. (a) Frames (200 × 200 nm) extracted from Movies S1–S3, taken respectively at 108 pN, 34 pN, or 14 pN and a fF of 0.56 min−1 at the indicated times. Horizontal scale bars are 50 nm. (b) Frames extracted from Movies S4–S6, were taken at a determined size and image points to keep constant the pixel size: 400 × 400 nm and 256 × 256 points for fF = 0.13 min−1; 200 × 200 nm and 128 × 128 points for fF = 0.48 min−1; 100 × 100 nm and 64 × 64 points for fF = 1.37 min−1 and 100 pN of applied force at the indicated times. For both (a) and (b), control (ctrl) images (right) taken at the end of each experiment cover a wider area to show that the area not subjected to mechanical fatigue was still fully coated, with no detectable disassembly even at the end of the experiment. The dark area at the center corresponds to the part that had been subjected to materials fatigue and fully disassembled under continual indentation. Horizontal scale bars for control images are 100 nm. To see this figure in color, go online. Biophysical Journal 2017 112, 663-673DOI: (10.1016/j.bpj.2016.11.3209) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 Kinetics of mechanically induced disassembly of the CA lattice on a solid substrate. (a) Substrate surface area covered as a function of time during disassembly of the CA lattice subjected to a fF of 0.56 min−1 under a force of 14 (diamonds), 34 (triangles), 55 (circles), or 108 (squares) pN. (b) Percent coverage as a function in time when the CA lattice were fatigued under a constant force of 100 pN and fF of 1.37 (squares), 0.48 (circles), or 0.13 (triangles) min-1. Data were fitted to sigmoidal curves (continuous lines). (c) Cooperative parameter (n) as a function of applied force. (d) n as a function of fF. (e) t50 value as a function of the applied force. (f) t50 value as a function of fF. In (e) and (f) continuous lines correspond to fittings to exponential decays. Error bars in (c) to (f) are derived from fitting errors of the data in (a) and (b) from which these parameters were obtained. To see this figure in color, go online. Biophysical Journal 2017 112, 663-673DOI: (10.1016/j.bpj.2016.11.3209) Copyright © 2017 Biophysical Society Terms and Conditions