RUB » RESOLV » EPR Spectroscopy
Bridge Q Band Marquard

@RUB, Marquard

Probing insertion and conformational transitions of proteins in lipid bilayers is a formidable task for any biophysical technique both in vivo and in vitro. Magnetic resonance techniques as liquid and solid state NMR can be combined to obtain information in the two tumbling regimes (from the aqueous solution to the membrane bilayer), but the bigger the size of the protein under study, the more challenging is following insertion into membranes or conformational changes. X-ray crystallography is one of the techniques which can give structural snapshots of membrane proteins in action in a membrane-like milieu (most crystals are obtained in detergent micelles), however, the challenges in this field are still numerous.

Site directed spin labeling (SDSL) EPR, in contrast, can be performed with the same sensitivity in both aqueous and lipid environment, thus allowing to follow insertion and conformational changes of membrane proteins in the same sample. Clearly, the drawback of this advantage is the need to genetically introduce cysteines, and subsequently EPR-active paramagnetic centers which give information at the molecular level on the proteins.

SDSL EPR techniques routinely used in the lab for protein science:

1. CW EPR at physiological temperature to obtain dynamics of the spin-labeled side chain
2. ODNP at physiological temperature to measure directly water accessibility towards a particular spin-labeld site, therefore topology of the protein with respect to the membrane-water interface
3. Dipolar spectroscopy, in particular DEER to extract interspin distances between spin-labeled sites, therefore intra- and inter-protein interactions. Orthogonal labels allow multiple interactions to be probed at once.