Synthesis of Modified and Labelled Lipids for Analysis of Enzyme Mechanisms and Membrane Interactions

by Christine Hansen
Doctoral thesis
Date of Examination:2017-10-09
Date of issue:2017-11-21
Advisor:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Ivo Feussner
Referee:Prof. Dr. Ebbe Nordlander
Persistent Address: http://dx.doi.org/10.53846/goediss-6590

 

 

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Abstract

English

Communication is key. The development of language represents a milestone in human evolution history. Long before verbal communication evolved, other, more essential forms of information transfer have arisen. The necessity of interaction and transduction of information is omnipresent in living systems. On extracellular and intracellular level communications take place via messenger compounds, which are divided into extracellular, first messenger and intracellular, second messenger. To investigate physiological processes, such as Ca2+ triggered neuronal exocytosis, an approach is the application of sensors. A major drawback of many dyeing techniques is the rapid exclusion of the fluorophore out of the cell. To prevent exclusion or compartmentalisation azide-carrying Ca2+ sensors were applied, which enabled coupling with alkyne containing compounds via 1,3-dipolar cycloaddition (HUISGEN or click reaction). The first project in this work is concerned with the conjunction of membrane compartments with such functionalised calcium sensors. Suchlike labelled molecules are then readily available for in vitro and in vivo experiments for Ca2+-dependent fluorescence measurements. Furthermore, SNARE-mediated synaptic vesicle fusion was to be studied while taking raft formation with increased amounts of PIP2 into account. Therefore, lipids were to be labelled with the well-studied 7-azaindole to monitor raft formation, since the fluorophore tends to form dimers in non-polar, aprotic surrounding, exhibiting a distinct red shift. In plants signalling agents are generated by conversion of fatty acids like jasmonic acid by LOXs. To investigate the substrate orientation within the active site of the enzyme 11,11-difluorolinoleic acid was to be synthesised. The geminal difluorinated fatty acid inhibits enzyme-driven conversion and enables co-crystallisation experiments of the substrate–enzyme complex.
Keywords: Lipid Modification; Calcium Sensor; Modified Fatty Acid; Ceramide; Fluorescence
 
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