Abstract
In this thesis a general concept for three-dimensional (3D) structure determination of uniformly [13C, 15N] labeled proteins by solid-state NMR spectroscopy has been developed. It is based on the combination of indirectly detected 1H-1H distance constraints and chemical shift derived backbone dihedral angle constraints. The collection of multiple distance constraints in two-dimensional CHHC and NHHC experiments has only recently been introduced. In order to include indirectly detected 1H-1H distance constraints in a molecular structure calculation, a comprehensive analysis of 1H-1H transfer in the solid-state was therefore mandatory. The concept was applied to the uniformly [13C, 15N] labeled 38-residue peptide kaliotoxin (KTX). Assigned CHHC correlations and chemical shift derived backbone dihedral angles (analyzed with the program TALOS) were used to determine a de novo 3D structure of KTX. CHHC data can also be used to obtain 3D structures of non-peptidic molecules. This was demonstrated on the [13C] labeled compound epothilone B, that binds with high affinity to polymerized tubulin. Finally the usage of high-resolution solid-state NMR spectroscopy to the investigation of high-affinity scorpion toxin / potassium channel interactions is described.
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