Skip to main content
Login | Suomeksi | På svenska | In English

Backbone NMR assignment of human activity-regulated cytoskeleton-associated protein C-lobe and analysis of a 7-fluoroindole-labeled sample using 1H15N chemical shift

Show full item record

Title: Backbone NMR assignment of human activity-regulated cytoskeleton-associated protein C-lobe and analysis of a 7-fluoroindole-labeled sample using 1H15N chemical shift
Author(s): Niemelä, Miska Aleksanteri
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences
Degree program: Master 's Programme in Neuroscience
Specialisation: Neuroscience
Language: English
Acceptance year: 2022
Abstract:
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.
Keyword(s): NMR Protein NMR nuclear magnetic resonance 7-fluoroindole protein expression structural biology


Files in this item

Files Size Format View
Niemela_Miska_Aleksanteri_thesis_2022.pdf 11.84Mb PDF

This item appears in the following Collection(s)

Show full item record