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Macromolecular Structure / Research Projects

Determination of membrane protein structures and conformational changes using site directed spin labelling and time resolved multi-frequency EPR spectroscopy

Our activities focus on the development of advanced electron paramagnetic resonance (EPR) methods, including site directed spin labelling, and their application to biomolecules, biomolecular complexes and nanoparticles. The interpretation of the experimental data is done in the frame of molecular dynamics simulation methods with the goal to better understand the structure-dynamics-function relationship of proteins and nucleic acids (see publications). Our research comprises studies of:

  • Light sensitive proteins:
    • The signal transfer mediated by the sensory rhodopsin - transducer complex
    • Light induced electron transfer in the bacterial photosynthetic reaction center
  • Transmembrane transporters:
    • ABC-transporters MalFGK2, Na/prolin transporter PutP, Gltph
  • Protein folding:
    • Colicin A and its interaction with the cell membrane with the perspective of in vivo application of site-directed spin labeling
  • Conformational dynamics of DNA and RNA

These activities are integrated in the Collaborative Research Center SFB 944.

Site directed spin labelling EPR spectroscopy of site directed spin labelled proteins is emerging as a promising tool to determine protein structures on the level of the backbone fold and to follow their conformational dynamics with time resolution in the millisecond regime. The introduction of nitroxide side chains is accomplished by cysteine substitution mutagenesis followed by reaction of the unique cysteine with a sulfhydryl specific nitroxide spin label. Information about the protein structure and its dynamics during protein function is extracted from the dynamics of the spin label side chain, from its accessibility to hydrophobic and hydrophilic paramagnetic quenchers (Pfeiffer et al., 1999), form the polarity of the binding site environment (Steinhoff et al., 2000b) and from distance measurements between two nitroxides in doubly spin labelled proteins (Steinhoff, 2004 and retated publications).

Side chain dynamics and molecular dynamics simulations

The spin label side chain dynamics is uniquely correlated to the secondary and tertiary structure in the environment of the nitroxide binding site. The EPR spectral shape is determined by the mechanism of the nitroxide residual motion (oscillation, restricted diffusion), its correlation time and its anisotropy, which contains the main information about the structure of the binding site. Multi-frequency EPR spectroscopy (9 GHz, 17 GHz, and 95 GHz) and EPR spectra calculations on the basis of molecular dynamics simulations allow to extract these parameters.

Spin-spin interaction and distance measurements

Tools to measure inter-residual distances are being developed and applied which are based on the evaluation of dipolar and exchange interaction between two nitroxide side chains or between a nitroxide side chain and a bound paramagnetic metal using pulse (DEER, PELDOR) or continuous wave EPR methods.


Over the last decades we applied site directed spin labelling and cw and time resolved EPR at X- and W- band to various retinal proteins (octopus rhodopsin, bacteriorhodopsin and sensory rhodopsin II). Besides insight into the physical principles of protein dynamics in general, we obtained detailed information about the structure and functional related conformational changes of these and other proteins (see publication list). They contribute to a better understanding on the mechanism of vectorial proton pumping in bacteriorhodopsin, reveal the pathway of signal transfer by the light receptor sensory rhodopsin, or show details of the conformational changes occuring during transmembrane transport.


Letzte Änderung: 13.12.2018 HJS