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Methods


Fakultät für Biologie und Vorklinische Medizin
Institute of Biochemistry, Genetics and Microbiology
Department of Structural Biology
Methods

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Methods

Our group is currently using a combination of structural biology, biochemistry, biophysics and functional studies to investigate the structure-function relationship of individual prokaryotic and eukaryotic membrane proteins as well as soluble protein complexes that affect transport regulation on transcriptional level.

 

1. Cryo-EM

The breakthrough of Cryo-EM technology has changed the landscape in Structural Biology field. Atomic resolution structures of many important large complexes and membrane proteins, unsolvable by x-ray so far have been determined since 2013 when direct electronic cameras and 3D reconstruction software initiated the “revolution of resolution”.

 

2. Crystallography

The amphiphilic nature of membrane proteins is a challenge in crystallization, because having both hydrophobic and hydrophilic portions membrane proteins will not readily dissolve into either a polar or an apolar solvent. Either the hydrophobic portions of the membrane protein will be shielded by a detergent micelle or like in meso method the membrane protein are hold in a membrane-like environment. Although less than 1% structures deposited in the Protein Databank are for membrane proteins solved by crystallization, X-ray diffraction is still important to obtain validated high-resolution atomic structures especially for human membrane proteins. We use it in combination with cryo-EM.

3. Membrane Protein Expression

We use baculovirus-mediated gene transfer into mammalian cells (BacMam) to express mammalian membrane proteins. BacMam can be used for large-scale protein production for crystallography and Cryo-EM. Membrane protein often show low levels of expression and instability depending on the constructs, which requires intensive screening. Furthermore, mammalian membrane proteins often require specific post-translational modifications and a near native lipid environment, which is only provided in mammalian cells.

 

4. Transport across membranes

We are using solid-supported membrane electro-physiology, fluorescence, ITC and MST as well as radioactive label substrate transport to characterize the transport of ions, nutrients.

 


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