Fig. 1.
Overview of experimental setup and src SH3 constructs studied. (A) Schematic diagram of the setup used to apply force to stalled RNCs using the optical tweezers. DNA handles are attached via an N-terminal Avi tag and a ybbr tag on protein L17 of the ribosome. (B) Constructs used in experiments for RNCs and free protein. SH3(38) and SH3(58) constructs contain linkers with 10 and 20 GS repeats, respectively. (C) Kinetic chevron plots of ln kobs against [urea] for src SH3 without the tags and linkers used in this work, free-SH3(38) and free-SH3(58). Data were collected in 25 mM Hepes, 150 mM KCl, 5 mM Mg-acetate, pH 7.4 (HKM) buffer. (D) Plots of ln kU against force for free-SH3(38) compared with previously published data with force applied in unzipping and shearing geometries. Unzipping and shearing data were collected in 100 mM Tris, 250 mM NaCl, pH 7, buffer, and free-SH3(38) were collected in HKM buffer.