A method for the genetically encoded incorporation of FRET pairs into proteins

by Christoph Lammers
Doctoral thesis
Date of Examination:2014-07-15
Date of issue:2014-09-03
Advisor:Prof. Dr. Heinz Neumann
Referee:Prof. Dr. Heinz Neumann
Referee:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Rolf Daniel
Referee:Dr. Fabian Moritz Commichau
Persistent Address: http://dx.doi.org/10.53846/goediss-4673

 

 

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Abstract

English

Proteins are composed of 20 canonical amino acids whose unique arrangements predefine a protein’s structure and function. Importantly, most proteins are not static conformations but rather very dynamic entities that undergo various structural modifications under different “stimuli”. The comprehension of these dynamic processes is necessary to understand how proteins work. Förster/Fluorescence resonance energy transfer (FRET) became a powerful tool to investigate conformational changes of proteins, and recent advances in technology haven given the capability for studies even on a single-molecule (sm) level. Therefore precise labeling of the proteins with suitable fluorophores is essential, however, remains a challenging task at present. Although chemical bioconjugation of fluorophores and proteins work, more or less reliably, specificity is a drawback for longer polypeptides and full-length proteins. To overcome the issue of specificity, synthetic biologists have opened new avenues by developing an expansion of the genetic code. This technique requires the introduction of exogenous nonsense suppressor tRNAs and their cognate aminoacyl-tRNA synthetases (aaRS) into the host cell, that have to work completely orthogonal to the endogenous components. This allows the incorporation of additional “unnatural” amino acids (UAAs) into proteins at the genetic level. These UAAs can bear many different functional groups with unique chemical or biophysical properties. Since we were interested in introducing two fluorophores, site-specifically into a protein, we had to use two tRNA/aaRS pairs, along with the plasmid harboring the gene of interest. This general approach necessitated multiple plasmids with different antibiotic resistances leading to heightened stress in the host cells. Additionally, using two non-cognate tRNA/aaRS pairs displayed toxic side-effects and required balanced levels within cells. Moreover, the two different UAAs drastically decreased the suppression efficiency and, in turn, the expression levels of protein. Therefore, an overall optimization of the system was essential. Herein, we describe the optimization process. We set out to reduce the number of plasmids used in this system, resulting in a highly modular genetic tool. We designed this system to allow for easy exchange with other tRNA/aaRS pairs to introduce new UAAs. We explored promoter libraries to fine-tune the expression levels of tRNA/aaRS pairs, which had profound effects on the UAA incorporation efficiency. Using the above system we achieved higher levels of protein expression with two different UAAs and are currently establishing bioorthogonal labeling strategies for use in smFRET studies.
Keywords: synthetic biology; amino acids; unnatural amino acids; genetic code expansion; FRET; click chemistry; system optimization; orthogonal ribosomes
 
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