Probing Small Molecule Binding to Unfolded Polyprotein Based on its Elasticity and Refolding  Ricksen S. Winardhi, Qingnan Tang, Jin Chen, Mingxi Yao, Jie Yan  Biophysical Journal  Volume 111, Issue 11, Pages 2349-2357 (December 2016) DOI: 10.1016/j.bpj.2016.10.031 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Schematic of the magnetic tweezers setup used to stretch and probe binding to unfolded polyprotein. Molecules that bind to the unfolded polyprotein may cause deformation to the unfolded polyprotein depending on the nature of binding. If a binding partner wraps unfolded polyprotein or promotes attractive interaction between remote residues, shorter extension upon binding is expected (scenario 2) as compared to the naked unfolded polyprotein (scenario 1). On the other hand, if binding leads to increased repulsive electrostatic or steric interactions between the residues, a longer extension is expected (scenario 3). Biophysical Journal 2016 111, 2349-2357DOI: (10.1016/j.bpj.2016.10.031) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Average force-height curves of a single dsDNA tether obtained from eight force-increase cycles. (Error bars) Standard deviation of bead height calculated from eight cycles of measurements. The acquisition time window is 2 s at a sampling rate of ∼200 Hz. (Inset) Intrinsic error normalized to a single amino acid as detailed in Measurement of Unfolded Polyprotein Elasticity (see main text). The normalized intrinsic error between two data points is approximated by linear interpolation. Biophysical Journal 2016 111, 2349-2357DOI: (10.1016/j.bpj.2016.10.031) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Force-extension data (per amino acid) obtained from six independent protein tethers. (Error bars) Intrinsic errors in Fig. 2 normalized to a single amino acid. Individual data sets are fitted with the WLC model, with a persistence length value of 1.00 ± 0.14 nm (mean ± SD). To see this figure in color, go online. Biophysical Journal 2016 111, 2349-2357DOI: (10.1016/j.bpj.2016.10.031) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 Effects of SDS binding on unfolded polyprotein force-extension curve. (A) Representative data obtained from a single protein tether at various concentrations of SDS in PBS buffer. Error bars represent intrinsic errors normalized to one amino acid. (Inset) Extension difference between an SDS-(unfolded-polyprotein) complex and an unfolded polyprotein at 6 pN obtained from multiple independent experiments. (B) Schematic diagram of the possible conformations adopted by SDS-(unfolded-polyprotein) complex at low concentration of SDS (scenario 2, wrapped mode), and high concentration of SDS (scenarios 3a and 3b, less wrapped micelle conformation and nonmicellar reorganization, respectively). To see this figure in color, go online. Biophysical Journal 2016 111, 2349-2357DOI: (10.1016/j.bpj.2016.10.031) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 Effects of SDS on (I27)8 refolding. (A) (Upper panel) Three force-increase time traces of extension obtained in three consecutive force cycles, each showing unfolding of (I27)8 domains indicated by characteristic stepwise extension increases. (Lower panel) Force values during the force-increase scans. (B) Representative unfolding time trace data obtained from a single protein tether. (Upper panel) Three force-increase time traces of the extension of an (I27)8 protein tether in the absence (black data, bottom) and in the presence of 0.01% (orange data, middle) and 0.1% SDS (blue data, top). (Lower panel) Force values during the force-increase scans. (C) Percentage of (I27)8 domains that were refolded in the absence and presence of SDS. The statistics was obtained from multiple force-cycle experiments of five independent protein tethers at each condition. The error bars represent standard deviations obtained from all the unfolding-refolding cycles. To see this figure in color, go online. Biophysical Journal 2016 111, 2349-2357DOI: (10.1016/j.bpj.2016.10.031) Copyright © 2016 Biophysical Society Terms and Conditions