Master's thesis project includes the backbone assignment of the human activity-regulated
cytoskeleton-associated protein C-lobe (hArc, Uniprot ID: Q7LC44), 7-fluoroindole-based
tryptophan-labeling method, and comparing that with the 100% double-labeled and 20%(13C)
fractionally labeled samples. The project focuses on the effects of 7-fluoroindole-based
fluorotryptophan-labeling. hArc C-lobe has only one tryptophan, which makes the analysis easier.
Typically fluorotryptophan-labeling is a costly method – fluorotryptophan itself is very expensive and
attaching the fluorine to the tryptophan while expressing is expensive and complicated. Fluoroindolebased
labeling circles around the problem, as indole and serine are used in procaryotic systems for
tryptophan biosynthesis – meaning that fluoroindole, which is cheap, could be used as an alternative
for previous methods.
Fluoro-labeled tryptophan is used in protein NMR; for example, in binding studies – fluorine-probes
are sensitive, and binding of ligand or protein would move these peaks, indicating binding. This
project aims to get an insight into the application of this labeling method. The goal is to see if one
could utilize one sample with both (1H, 15N, 13C) labeling and 7-fluorotryptophan labeling for binding
and structural studies. However, fluorine is very electronegative, affecting surrounding structures and
possibly sequentially nearby amino acids. This possible effect will be observed and determined by
comparing the 1H15N-chemical shifts between well-established labeling methods and fluoroindolebased
labeling. To determine what amino acids in the protein are affected, if they are affected, will be
determined by using the backbone assignment results and the results from the sample comparisons.
