WP1: protein engineering
Magnetic field-activated channel: We have analyzed their iron content of the ferritins and, together with our collaborators from WP3, tested by NMR, confocal microscopy and atomic force spectroscopy their properties. So far, our conclusions are: 1) human and bacteria ferritins assemble correctly into 24-mers; 2) the amount of iron accumulated by human ferritin in HEK293 cells is very low compared to what reported in the literature; 3) in our experimental conditions, they do not seem to respond to changes in magnetic field. In the future, we plan to increase iron uptake into HEK 293 cells by supplying external iron and/or by expressing an iron transporter.
Far red-activated channel: bacterial phytochrome protein were circularly permutated in three positions. We are testing the new constructs in yeast cells, by confocal microscopy and spectroscopy if they fold correctly. The next step will be to connect them to the potassium channel TREEK.
Temperature-dependent channel: We have prepared two prototypes that were tested by patch clamp. They are respectively fully closed and partially closed at room temperature (25 °C). The first one was tested at higher temperature, 45 °C, showing recovery of activity.
Ultrasounds-regulated channel: we have selected a viral channel with maximal exposure to the lipid bilayer. This channel has been characterized in artificial lipid bilayer and has been purified from the yeast Pichia pastoris cells as well as in-vitro transcribed and translated in a cell-free system.
Blue-light regulation of K+ channel: we have improved the mammalian expression of our previous engineered channel BLINK. The new channel, BLINK2, was obtained by adding the regulatory sequence for 14-3-3 binding identified in the plant channel KAT1 (see publication Saponaro et al., 2017, Plant Cell). BLINK2 was tested in ex vivo in mouse brain and in vivo in a rat model for chemiotherapy-induced neuropathic pain.
Blue-light regulation of ion channel expression by Cryptochrome: To widen the scope of light activated channel systems we employed a strategy which allows a functional expression of channels in the plasma membrane or in the mitochondria in a light regulated manner.
WP2: computational approaches
We have developed computational methods, which provide insights into the mechanical connectivity of ion channels. The application of anisotropic network models and a more refined application of the linear responds theory (LRT) to ion channel proteins have paved the way for understanding long range interactions and conformational changes in channel proteins.
Protein/lipid interactions: To understand this lipid/protein interaction we have investigated the responds of small model K+ channels in variable lipid environment. To this end we developed a new technique, which combines in vitro protein translation and nanodisc technology, for functional reconstitution of channel proteins into bilayers with variable compositions.
WP3: atomic force microscopy (AFM), NMR and SAXS approaches
In the frame of WP3, during these months, we performed atomic force microscopy (AFM) imaging on several ferritin samples. The experiments were performed in Peak Force Tapping mode in fluid, a technique which provides very accurate control of the applied forces.
Nanodiscs: we performed fluid AFM imaging in Peak Force Tapping of nanodiscs containing a tetrameric channel, both on bare mica and after fusion in a supported phospholipid bilayer. Data analysis show the presence of particles with a height of about 4.5 nm.
NMR, nuclear magnetic resonancewe performed H1 NMR of Pyrococcus furiosus holo-ferritin with a spectrophotometer FT-NMR, 600 MHz, Bruker. The Buffer signals were too intense compared to those of the sample sample, so it was not possible to obtain information with this technique.
SAXS, small angle X-ray scattering
A proper protocol for efficient and safe channel delivery to a model membrane has been developed and structural interference between the Kcv channel and the hosting membrane has been investigated by calorimetry and X-ray and neutron spectroscopy.
