Unfolding Dynamics of the Cyclic Nucleotide Binding Domain and C-Linker of HCN Channels

Abstract

The cyclic nucleotide binding (CNB) domain of a variety of proteins is composed by a binding pocket formed by several alpha helices and beta sheets. In HCN channels, the CNB domain is connected to the C-linker, forming the cytoplasmic domain that modulates channel gating. We have performed single molecule force spectroscopy experiments on the hHCN4 construct formed by 203 aminoacids spanning the C-linker and the CNB domain (from aa 521 to aa 723). The construct was deposited on (3-Mercaptopropyl)trimethoxysilane (MPTs) functionalized mica surfaces and we collected Force-distance (F-d) traces in the absence and in the presence of 2mM cAMP. In the presence of cAMP force peaks at contour length (Lc) of 33.4±1.5, 43±1.5 and 52.5±2nm were detected with forces between 45 and 64pN, and at longer values of Lc corresponding to 63.8±2.5 and 84.5±1.5 nm with forces between 86 and 107pN. In the absence of cAMP force peaks at Lc of 34±1.5, 59.5±2 and 65±1.5 are detected with forces between 46 and 52pN, and at Lc of 74±1.5 and 86±2.5 with forces between 80 and 106pN. As alpha helices unfold at lower forces than beta sheets, these results suggest that F-d traces up to a Lc of about 55nm are associated to the unfolding of alpha helices and from 55 to 90nm to the unfolding of beta sheets. The force peak with a value of Lc around 43nm observed in the presence of cAMP is not seen in the absence of cAMP suggesting that several alpha helices are not properly folded in the absence of cAMP, to a greater extent than previously thought (Taraska et al. 2009). These results suggest also that the beta sheet has a different folding than that in the absence of cAMP.