All posts by : Karin Janz

Cryogenic electron microscopy (cryo-EM) has become the preferred method for determining the atomic-resolution structures of macromolecules ¹. The preparation of cryo-EM specimens is a multi-step procedure, typically divided into four main stages: (i) glow-discharging the cryo-EM grids, (ii) applying the macromolecular sample to the grid, (iii) blotting off excess protein solution, and (iv) vitrifying the specimen in a cryogenic liquid. Specialized equipment is employed in steps (iii) and (iv) to meticulously control these processes. The importance of vitrification in cryo-EM grid preparation was highlighted when the Swiss researcher Jacques Dubochet received the Nobel Prize in Chemistry in 2017 for his pioneering work in this area (https://www.nobelprize.org/prizes/chemistry/2017/summary/).

Cryo-EM grids used for high-resolution protein structure determination are typically made of copper or gold with a holey carbon film attached on one side. Chemically, this surface is hydrophobic and would naturally repel hydrophilic protein samples. Therefore, cryo-EM grids must be rendered hydrophilic through a process called glow-discharge. Since the glow-discharging procedure significantly impacts protein absorption behavior on cryo-EM grids, precise control of this process is essential. With the funding acquired through the UniBern Forschungsstiftungs grant, we obtained a Dual-Chamber PELCO easiGlow™ glow-discharging device, which allows for controlled glow-discharging and ensures a more reliable and reproducible production of cryo-EM specimens (Figure 1A).

Protein synthesis is essential for any living organism. However, how the dynamics of mRNA translation affect the formation of functional proteins is poorly understood. To gain new insights into the relationship between translation dynamics and protein folding, we analyze mutants that show codon-specific translation defects.

The goal of our project is to develop a platform that allows the identification of small molecules that inhibit translation in human cells using cell-free lysates. The lysates are also used for studying the mode of action of proteins from coronaviruses (2,3).

Numerous human diseases are caused by genetic or acquired ion channel dysfunction called channelopathies. Ion channels are membrane proteins allowing the passage of ions. The cardiac sodium ion channel Na_v1.5, which gene is SCN5A, plays a pivotal role in such disorders. Na_v1.5 channels are mainly expressed in the heart. Several hundred genetic variants in SCN5A were found in patients with a broad spectrum of cardiac manifestations, such as LQTS type 3, BrS, atrial fibrillation, and dilated cardiomyopathy. As with other proteins, the Na_v1.5 channel interacts with many proteins, leading to its fine-tuning of expression, localization, and function. The work of our group is focused on deciphering which partner proteins interact with Na_v1.5, leading to its proper function. The main technics used to perform such investigations are biochemical assays named co-immunoprecipitation (co-IP) experiments. However, the crucial step for this biochemical assay is the extraction of large proteins such as Na_v1.5 α-subunit monomer (~220 KDa). In addition to their size, the embedment of those ion channels in a lipid bilayer, such as a plasma membrane, renders the extraction more difficult than cyto-soluble protein.

The homogenizer ‘Bioprep-24R’’ (ALLSHENG) that we were able to acquire, thanks to the UniBern Forschungsstiftungs’ grant, becomes essential for such investigation. The team members largely benefit from this new equipment to extract efficiently voltage-gated ion channels expressed in different organs and tissues. The homogenizer permits fast, effective, and reproducible homogenization, relating to the three-dimensional high-speed vibration and beating of grinding beads (glass beads, ceramic beads, steel balls, etc.).

We can now efficiently extract Na_v1.5 channels from animal organs, a critical step to performing co-immunoprecipitation experiments.

PD Dr. Jean-Sébastien ROUGIER
Institute of Biochemistry and Molecular Medicine

Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions.