Methods

Our research group employs a multidisciplinary approach combining structural biology, biochemistry, biophysics and functional studies to investigate the relationship between the structure and function of both prokaryotic and eukaryotic membrane proteins. Our focus extends to soluble protein complexes that impact the regulation of transport at the transcriptional level.

1. Cryo-Electron Microscopy (Cryo-EM):
The advent of Cryo-EM has revolutionized the field of Structural Biology. This technique has enabled the determination of atomic resolution structures for large complexes and membrane proteins that were previously inaccessible to X-ray crystallography. Since the introduction of direct electron cameras and advanced 3D reconstruction software in 2013, Cryo-EM has significantly enhanced our understanding of these macromolecules.

2. X-ray Crystallography:
Although membrane proteins present challenges in crystallization due to their amphiphilic nature, X-ray diffraction remains a valuable tool to obtain high-resolution atomic structures, particularly for human membrane proteins. We combine this method with Cryo-EM to gain comprehensive insights into membrane protein structure and function. Detergent micelles or the meso method, providing a membrane-like environment, are used to solubilize the hydrophobic portions of these proteins.

3. Membrane Protein Expression:
To express mammalian membrane proteins, we employ the baculovirus-mediated gene transfer into mammalian cells (BacMam) system. This allows for the large-scale production of these proteins suitable for crystallography and Cryo-EM. Mammalian membrane proteins often exhibit low expression levels and instability, necessitating intensive screening. Additionally, specific post-translational modifications and a near-native lipid environment can only be provided within mammalian cells.

4. Transport Across Membranes:
Our investigations employ multiple techniques, including solid-supported membrane electrophysiology (SSM), fluorescence, isothermal titration calorimetry (ITC), microscale thermophoresis (MST), and radioactive substrate transport assays to characterize the transport of ions and nutrients across membranes. These methods provide valuable insights into the mechanisms and kinetics of transport processes.

By employing this integrative methodology, we aim to unravel the complexities of membrane protein function and contribute to a deeper understanding of cellular transport systems, ultimately paving the way for potential applications in various fields, including clinical research.