Fluorescent Protein Toolbox: Protein Engineering Broadens the Range of in vitro and in vivo Applications of Fluorescent Proteins
George Abraham, Bobin (2015)
Tampere University of Technology
2015
Luonnontieteiden ja ympäristötekniikan tiedekunta - Faculty of Science and Environmental Engineering
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Julkaisun pysyvä osoite on
https://urn.fi/URN:ISBN:978-952-15-3655-7
https://urn.fi/URN:ISBN:978-952-15-3655-7
Tiivistelmä
In the last two decades, fluorescent proteins have become one of the most widely studied and exploited protein in biochemistry and cell biology. Fluorescent protein is a protein that upon excitation at low wavelength light emits fluorescence at higher wavelength. Its ability to generate high intracellular visibility together with the stable internal fluorophore and non-invasive measurement technologies made it the finest tool to monitor cellular processes and molecular events in living cells at its normal physiological conditions. Protein engineering and identification of novel fluorescent proteins have resulted in the development of color variants ranging from the blue to near-infrared region of the spectrum. Protein engineering has also lead to the development of highly stable fluorescent proteins with improved photochemical properties and sensing abilities.
The fluorescent proteins have made a strong impact in cell biology research due to its ability to participate in energy transfer interactions, such as Fluorescence resonance energy transfer (FRET) and thus allowing to measure and study molecular-scale distances and dynamics through changes in fluorescence. Development of novel FRET based techniques, FRET sensors and FRET pairs will provide opportunity to understand the cellular processes and dynamics with high precision at nano-scale level. This thesis focusses on FRET studies by developing novel FRET based sensor, novel FRET pairs and analyzing intramolecular FRET. The study also focuses on analyzing the potential of fluorescent proteins in sensing applications outside the cell environment, an area which has not yet been exploited. This was accomplished by protein engineering of fluorescent proteins with specific objectives followed by steady-state and time-resolved fluorescence spectroscopy measurements.
In one of the specific objective, intramolecular FRET in fluorescent proteins was studied by demonstrating FRET between fluorescent protein and conjugated chemical fluorophores whereby FRET occurs from inside to outside of the protein and vice versa. For this study, novel FRET pairs MDCC−Citrine and Citrine− Alexafluor 568 was generated. FRET analyzed using steady-state and ultra-fast time-resolved spectroscopy measurements revealed strong intramolecular FRET with high efficiencies. To my knowledge, this is the first and only study on bidirectional FRET between fluorescent protein and conjugated chemical labels. This study was made possible by genetically engineering Citrine to incorporate cysteine residues on the surface of the protein and this enabled site-specific bioconjugation of the labels to the fluorescent protein.
The surface exposed cysteine on the fluorescent protein was also exploited in this study to generate self-assembled monolayer (SAM) of Citrine on the surface of etched optical fibers (EOF). The conjugation of Citrine to the surface of EOF demonstrated a proof-of-concept for the use of this bio-conjugated protein in in vitro bio-sensing applications. To the best of our knowledge, this is the first and only study on the formation of fluorescent protein SAM on EOF. Steady-state and fluorescence lifetime measurements confirm the formation of SAM on EOF and revealed that the bioconjugation is site-specific and covalent in nature. The study also demonstrates that the proteins retains its photochemical properties on bioconjugation and are stable at physiological conditions.
The engineered surface exposed cysteine was further used in this study for the development of a FRET based redox sensor. This was developed aiming to overcome the disadvantages of the current FRET based redox sensors which includes low FRET efficiency and dynamic range, and to monitor the redox status in bacteria. For the sensor development, fluorescent proteins Citrine and Cerulean were genetically engineered to expose reactive cysteine residues on the protein surface. The proteins were fused using a biotinylation domain as a linker to generate the FRET sensor. The redox titrations and the fluorescence measurements confirmed the redox response and reversibility of the sensor. The FRET sensor exhibited high FRET efficiency and dynamic range in intensity based measurements. Intracellular studies with Escherichia coli revealed the capability of the FRET sensor in detecting real-time redox variations at single cell level.
In the final study, novel FRET pairs were developed aiming at improved fluorescence lifetime dynamic range and high FRET efficiency for the use in fluorescence lifetime imaging microscopy (FLIM) studies. The fluorescent protein with the longest reported fluorescence lifetime NowGFP was used as a FRET donor and various red-fluorescent protein variants were screened for the optimal FRET acceptor. Among the FRET pairs screened, NowGFP-tdTomato and NowGFP-mRuby2 were found to be superior FRET pairs with high lifetime dynamic range and FRET efficiency. NowGFP-tdTomato pair was found to have the highest reported Förster radius and fluorescence lifetime dynamic range for any fluorescent protein based FRET pairs yet used in biological studies.
In summary, we have developed novel FRET based tools and in vitro techniques using fluorescent proteins which can assist in deepening the knowledge on intracellular environment and dynamics, and also in developing novel fluorescent protein based sensors which can be used outside the cellular environment.
The fluorescent proteins have made a strong impact in cell biology research due to its ability to participate in energy transfer interactions, such as Fluorescence resonance energy transfer (FRET) and thus allowing to measure and study molecular-scale distances and dynamics through changes in fluorescence. Development of novel FRET based techniques, FRET sensors and FRET pairs will provide opportunity to understand the cellular processes and dynamics with high precision at nano-scale level. This thesis focusses on FRET studies by developing novel FRET based sensor, novel FRET pairs and analyzing intramolecular FRET. The study also focuses on analyzing the potential of fluorescent proteins in sensing applications outside the cell environment, an area which has not yet been exploited. This was accomplished by protein engineering of fluorescent proteins with specific objectives followed by steady-state and time-resolved fluorescence spectroscopy measurements.
In one of the specific objective, intramolecular FRET in fluorescent proteins was studied by demonstrating FRET between fluorescent protein and conjugated chemical fluorophores whereby FRET occurs from inside to outside of the protein and vice versa. For this study, novel FRET pairs MDCC−Citrine and Citrine− Alexafluor 568 was generated. FRET analyzed using steady-state and ultra-fast time-resolved spectroscopy measurements revealed strong intramolecular FRET with high efficiencies. To my knowledge, this is the first and only study on bidirectional FRET between fluorescent protein and conjugated chemical labels. This study was made possible by genetically engineering Citrine to incorporate cysteine residues on the surface of the protein and this enabled site-specific bioconjugation of the labels to the fluorescent protein.
The surface exposed cysteine on the fluorescent protein was also exploited in this study to generate self-assembled monolayer (SAM) of Citrine on the surface of etched optical fibers (EOF). The conjugation of Citrine to the surface of EOF demonstrated a proof-of-concept for the use of this bio-conjugated protein in in vitro bio-sensing applications. To the best of our knowledge, this is the first and only study on the formation of fluorescent protein SAM on EOF. Steady-state and fluorescence lifetime measurements confirm the formation of SAM on EOF and revealed that the bioconjugation is site-specific and covalent in nature. The study also demonstrates that the proteins retains its photochemical properties on bioconjugation and are stable at physiological conditions.
The engineered surface exposed cysteine was further used in this study for the development of a FRET based redox sensor. This was developed aiming to overcome the disadvantages of the current FRET based redox sensors which includes low FRET efficiency and dynamic range, and to monitor the redox status in bacteria. For the sensor development, fluorescent proteins Citrine and Cerulean were genetically engineered to expose reactive cysteine residues on the protein surface. The proteins were fused using a biotinylation domain as a linker to generate the FRET sensor. The redox titrations and the fluorescence measurements confirmed the redox response and reversibility of the sensor. The FRET sensor exhibited high FRET efficiency and dynamic range in intensity based measurements. Intracellular studies with Escherichia coli revealed the capability of the FRET sensor in detecting real-time redox variations at single cell level.
In the final study, novel FRET pairs were developed aiming at improved fluorescence lifetime dynamic range and high FRET efficiency for the use in fluorescence lifetime imaging microscopy (FLIM) studies. The fluorescent protein with the longest reported fluorescence lifetime NowGFP was used as a FRET donor and various red-fluorescent protein variants were screened for the optimal FRET acceptor. Among the FRET pairs screened, NowGFP-tdTomato and NowGFP-mRuby2 were found to be superior FRET pairs with high lifetime dynamic range and FRET efficiency. NowGFP-tdTomato pair was found to have the highest reported Förster radius and fluorescence lifetime dynamic range for any fluorescent protein based FRET pairs yet used in biological studies.
In summary, we have developed novel FRET based tools and in vitro techniques using fluorescent proteins which can assist in deepening the knowledge on intracellular environment and dynamics, and also in developing novel fluorescent protein based sensors which can be used outside the cellular environment.
Kokoelmat
- Väitöskirjat [4901]