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Abstract

The problem of structure determination of membrane proteins is addressed with a new combination of
site-directed spin labelling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and structure
modelling of a protein and its conformational spaces. This new approach is aimed at structural
characterization of membrane proteins and intrinsically disordered proteins.
In the first part of the thesis the advanced analysis of EPR spectra based on spectral simulations and
condensation of multiple solutions was enhanced facilitating its application for protein structure
characterization. In the second part a novel approach was developed to simulate the free rotational space of
a spin label attached to a protein, taking into account the restricting effect of the protein backbone, amino
acid side chains and lipid environment in case of membrane proteins. In the third part this simulations were
coupled with protein backbone modelling and an optimization algorithm to optimize the secondary
structure of the protein as well as the parameters of relative position and orientation in a protein-lipid or
protein-protein systems. The outcome of the optimization is a family of best-fit structures used for
characterization of the global conformation of a protein. Finally, the method was applied to study the
structure of the membrane-embedded major coat protein of bacteriophage M13 as well as the intrinsically
disordered NTAIL protein in NTAIL-XD protein complex of the measles virus.
Thus, the present method represents a challenging starting point for the development of an alternative
powerful methodology for the structure characterization of proteins, particularly intrinsically disordered or
membrane proteins as the combination of EPR and structural modelling provides a valuable complement to
the time and spatial windows of the conventional techniques used in protein structure determination.