Non-Canonical Amino Acids as Minimal Tags for Investigating Protein Organization and Turnover

by Ingrid-Cristiana Gebura-Vreja
Doctoral thesis
Date of Examination:2015-10-14
Date of issue:2016-01-08
Advisor:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Blanche Schwappach
Persistent Address: http://dx.doi.org/10.53846/goediss-5450

 

 

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Abstract

English

The burgeoning field of genetic code expansion provides new tools for specifically labeling proteins for a variety of applications. Clickable non-canonical amino acids (ncAA) have been refined for almost-quantitative and highly selective reactions with complementary probes. In my thesis work I have adapted genetic code expansion for two biological questions. First, I used ncAA incorporation and click reaction to test the effect of fluorescent protein (FP) tagging on the nanoscale organization of target proteins. Second, I used these tools to generate a genetically-encoded scheme for specific protein labeling in non-optical nanoscale imaging. Protein localization and behavior has been regularly tested using FPs. Conventional imaging experiments using FPs are simple and efficient, which renders FPs attractive also for super-resolution microscopy (nanoscopy). Nevertheless, FPs have been claimed to induce the nanoscale aggregation of target proteins. Therefore, the effects of FP-tagging on the nanoscale organization behavior of the target proteins needed to be tested, in an unbiased fashion, using a reporter that is smaller and less artifact-prone than the FPs. I relied on the specific incorporation of the ncAA propargyl-L-lysine (PRK) into the FP chimeras of 26 proteins of interest, both cytosolic and membrane attached. The proteins were coupled via click chemistry to fluorescent probes suitable for either stimulated emission depletion microscopy (STED) or ground state depletion followed by individual molecule return (GSDIM). Analysis of the resulting images showed that FP tagging has negligible effects on most proteins, and therefore supported the use of FPs in nanoscale imaging. Optical microscopy is not the only nanoscale imaging approach that can be used at the moment. Nanoscale secondary mass spectrometry (NanoSIMS) relies on isotope measurements to reach a similar resolution domain. However, it lacks genetically encoded, FP-like tools. For this purpose I developed a novel labeling scheme, for specific protein isotopic and fluorescence labeling (SPILL). It involves the incorporation of ncAA PRK followed by the reaction with two novel probes containing isotopes that are not normally abundant in cells, 19F and 15N. These isotopic probes can be imaged in SIMS akin to GFP in optical microscopy, and enable the high resolution imaging of many cellular parameters, including protein turnover.
Keywords: non-canocial amino acids, protein engineering, super-resolution microscopy, secondary ion mass spectrometry
 
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