RESUMEN
Structural maintenance of chromosome (SMC) complexes form ring-like structures through exceptional elongated coiled-coils (CCs). Recent studies found that variable CC conformations, including open and collapsed forms, which might result from discontinuities in the CC, facilitate the diverse functions of SMCs in DNA organization. However, a detailed description of the SMC CC architecture is still missing. Here, we study the structural composition and mechanical properties of SMC proteins with optical tweezers unfolding experiments using the isolated Psm3 CC as a model system. We find a comparatively unstable protein with three unzipping intermediates, which we could directly assign to CC features by crosslinking experiments and state-of-the-art prediction software. Particularly, the CC elbow is shown to be a flexible, potentially non-structured feature, which divides the CC into sections, induces a pairing shift from one CC strand to the other and could facilitate large-scale conformational changes, most likely via thermal fluctuations of the flanking CC sections. A replacement of the elbow amino acids hinders folding of the consecutive CC region and frequently leads to non-native misalignments, revealing the elbow as a guide for proper folding. Additional in vivo manipulation of the elbow flexibility resulted in impaired cohesin complexes, which directly link the sensitive CC architecture to the biological function of cohesin.
RESUMEN
Single-molecule force spectroscopy using optical tweezers continues to provide detailed insights into the behavior of nanoscale systems. Obtaining precise measurements of their mechanical properties is highly dependent on accurate instrument calibration. Therefore, instrumental drift or inaccurate calibration may prevent reaching an accuracy at the theoretical limit and may lead to incorrect conclusions. Commonly encountered sources of error include inaccuracies in the detector sensitivity and trap stiffness and neglecting the non-harmonicity of an optical trap at higher forces. Here, we first quantify the impact of these artifacts on force-extension data and find that a small deviation of the calibration parameters can already have a significant downstream effect. We then develop a method to identify and remove said artifacts based on differences in the theoretical and measured noise of bead fluctuations. By applying our procedure to both simulated and experimental data, we can show how effects due to miscalibration and trap non-linearities can be successfully removed. Most importantly, this correction can be performed post-measurement and could be adapted for data acquired using any force spectroscopy technique.
