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Spectroscopy in cells with tailored in-vivo labelling strategies and multiply addressable nano-structural probes

Periodic Reporting for period 3 - SPICE (Spectroscopy in cells with tailored in-vivo labelling strategies and multiply addressable nano-structural probes)

Berichtszeitraum: 2021-06-01 bis 2022-11-30

The native structure of proteins in living cells has remained largely unexplored owing to the lack of suitable structure determination methods. Prof Malte Drescher and his interdisciplinary research team at the University of Konstanz, Germany, develops novel spectroscopic approaches to investigate structure and dynamics of proteins in living cells. These methods are an important link between classical molecular biophysics and systems biology. Because of their high potential, electron paramagnetic resonance (EPR) spectroscopy techniques in combination with site-directed spin labelling are of particular interest.
Intrinsically disordered proteins (IDPs), implicated in human diseases, among them prominently cancers, cardiovascular diseases, diabetes and neurodegenerative diseases, adopt a rich variety of different conformations depending on the macromolecular context. In order to unravel their pathophysiological role, monitoring their intracellular conformational states and identifying differences for the disease variants is crucial.
Therefore, the Drescher group pushes the experimental conditions from in vitro experiments towards structure determination in vivo.
The group investigated, how molecular labels can be activated, i.e. switched to a magnetic state, using a laser beam. Measuring the strength of their magnetic interaction by electron paramagnetic resonance spectroscopy, they can conclude on their distance in the naometre range. Therefore, it is useful to attach them to proteins in order to obtain distance restraints.
Therefore, the team incorporated genetically encoded, artificial amino acids into proteins and used them as targets for their labels.
For the first time, they combined in-cell labeling and in-cell EPR spectroscopy. The combination of intracellular bioorthogonal labeling with in-cell EPR measurements does not require additional purification or delivery steps of spin-labeled protein to the cells.
The Drescher group also applied EPR spectroscopy to study aggregation of the microtubule-associated protein Tau, which is a hallmark of Alzheimer’s disease. They found that the interaction with Hsp90 promotes an open Tau conformation, which they identified as the molecular basis for the formation of small Tau oligomers.
The Drescher group for the first time applied rapid scan EPR spectroscopy for the first time in biological cells. Rapid scan EPR is a rapidly emerging technology and enables significantly higher signal to noise ratio and / or time resolution with respect to conventional EPR spectroscopy. The protein under study – alpha-Synuclein – is an intrinsically disordered protein and of high relevance as model system as well as "Parkinson protein". The experiments revealed for the first time the behavior of alpha-Synuclein in terms of membrane interactions within cells.