Mechanical unfolding of the cytosolic domain from hHCN2 channel by using single molecule force spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS) has emerged into a reliable technique for probing structural and mechanical properties of biological samples at molecular level. It has been successfully applied to investigate the mechanical unfolding of soluble proteins as well as membrane proteins. In contrast with the traditional structural techniques it provides a direct mechanical interaction with targets molecule in their near-physiological environment. The possibility to sample one molecule at a time, made the techniques extremely valuable for studying complex dynamic behaviors, unveiling rare events that are usually averaged in large data distribution from big populations of molecules. Here we investigate the mechanical unfolding of the purified cytosolic c-linker and cyclic nucelotide binding domain (CNBD) domain from the human hyperpolarization-activated and cyclic nucleotide-gated (hHCN)2 channel, and characterize the ligand-depended differences in the unfolding behaviour of the molecule. In parallel, we develop an all-in-one environment for Force vs distance (F-d) curves analysis, containing informatics tools to handle the reproducibility and automation required for exploring stochastic processes like folding and unfolding of complex proteins.