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

Project Information

SPICE

Grant agreement ID: 772027

DOI

10.3030/772027

Project closed

EC signature date 18 April 2018

Start date 1 June 2018

End date 30 November 2024

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 988 697,50

EU contribution

€ 1 988 697,50

Coordinated by

UNIVERSITAT KONSTANZ
Germany

Periodic Reporting for period 5 - SPICE (Spectroscopy in cells with tailored in-vivo labelling strategies and multiply addressable nano-structural probes)

Reporting period: 2023-12-01 to 2024-11-30

Summary of the context and overall objectives of the project

The native structure of proteins in living cells has remained largely unexplored owing to the lack of suitable structure determination methods. The interdisciplinary SPICE research team at the University of Konstanz, Germany, developed 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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The SPICE team designed and established molecular proebes, i.e. labels for experiments which can be addressed via several channels, i.e. different spectroscopic approaches.
These multiply-addressable nano-structural probes give access to a large set of length scales using the very same sample.

The SPICE team developed tailored strategies for labelling in vivo, mainly based on genetically encoded non-canonical amino acids.

Knowledge and technology transfer: Cancer is a global public health problem. Radiation therapy (radiotherapy) is generally used as an essential method of cancer treatment to slow down or control tumor growth by killing malignant cells while spare healthy tissue.
Measuring the absorbed dose of radiation to ensure that the radiation delivered to the patient is accurate, is paramount and critical to patient safety, since tumor control and side-effects are highly sensitive to the applied dose. For this purpose, in-vivo dosimetry can be performed as a patient-specific measure of quality control and safety during radiotherapy. Water equivalent dosimeters are not available but particularly desirable in clinical applications. OThe SPICE team proposed a small, water-equivalent, non-toxic and liquid dosimeter with electron paramagnetic resonance spectroscopy readout which is further developed within the proof of concept grant LIQUIDITY.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The researchers pushed the domain of molecular spectroscopy for structural biology and biophysics into the living cell. For the first time, they combined in-cell labeling and in-cell EPR dipolar spectroscopy. Their approach does not require additional purification or delivery steps of spin-labeled protein to the cells and allows for in-cell EPR distance determination in the nanometer range.

The SPICE team also applied EPR spectroscopy to highly relevant protein systems.

As a prominent example, they identified the interaction of the tau protein (associated with Alzheimer’s disease) with another protein named Hsp90 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.

Another 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.

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