Single Molecule Force Spectroscopy of TMEM16F and SthK channels

The cell membrane proteins act as the gates between the extracellular world and the intracellular part of cells, mediating signals communication between the internal and external environment of cells. These processes are fundamental for the survival of cells. Their abnormal functions cause many kinds of diseases. What’s more, membrane proteins are the major targets of the current available drugs. But still, we human beings can’ t cure all kinds of diseases, because of the unknown-structural information of membrane proteins. It is well established that nowadays the most popular way to acquire the atomic structures is by the means of cryo-electron microscopy (cryo-EM) and x-ray crystallography. These methodologies provide a detailed description of the whole structures which help for understanding how these molecules operate in physiological conditions and in disease, ultimately leading to better drugs’ design with fewer unwanted side effects. However, these structural techniques have their limitations. Firstly, Cryo-EM needs to extract the target molecules from cells and to reconstitute them in nanodisc, to put them into the suitable solutions and to freeze and in these processes the target molecules lose their native states and regulatory associated co-factors. During these processes, it’s hard to maintain molecule’s structural and functional integrity and therefore wrong structural determination cannot be avoided. Secondly, all these manipulations are made at very low temperature, commonly at 4 °C, which is very different from the normal environment (normal temperature and pressure) that is suitable for our organism. Thirdly, they make use of over tens of thousands of molecules and then obtain near-atomic resolutions by averaging them. Therefore, losing the multitude of structural conformations expected in a large and complex molecule. This is in sharp contrast with atomic force microscopy (AFM) which can directly probes a single molecule at a time. The AFM technology has two major applications: AFM-based single molecule force spectroscopy (SMFS) and High-Speed AFM (HS-AFM). AFM-SMFS can manipulate and investigate the mechanical properties of single molecules via its unequivocal sensitivity while HS-AFM is able to film single molecules at high spatiotemporal resolution. What is important and at the basis of my PhD thesis is that both SMFS and HS-AFM work at room temperature and can handle samples in their native environment. In my thesis, I have combined different technologies, such as AFM, HS-AFM, electrophysiology and simulation, to probe the dynamic conformations of TMEM16F and SthK channels. My results show that: 1. The molecular structure of the TMEM16F membrane protein is highly dynamic. The TMEM16F is a dimer protein with each subunit bearing different conformations and the interface of the two subunits is much more dynamic than expected. In addition, I have been able to see the scramblase activity – a peculiar property of TMEM16F channels – at a single molecule level by using HS-AFM. 2. SthK channel is highly heterogeneous. SthK molecules dynamically blink (switch back and forth) between different conformations with different heights. The cyclic nucleotide-binding domain even in the presence of cAMP, an agonist of SthK, does not have a well folded configuration which in the absence of cAMP has a more “loose” conformation compared with that of the cAMP-bound SthK. 3. These results show that the molecular structures obtained by Cryo-EM must be complemented by approaches working at the single molecule level such as SMFS and HS-AFM.