Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor
Isoleucine is one of the branched chain amino acids that plays a major role in the energy metabolism of human beings and animals. However, detailed investigation of specific receptors for isoleucine has not been carried out because of the non-availability of a tool that can monitor the metabolic flu...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2019-12-01
|
| Series: | Sensors |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1424-8220/20/1/146 |
| _version_ | 1818000921551962112 |
|---|---|
| author | Shruti Singh Maheshwar Prasad Sharma Abdulaziz A. Alqarawi Abeer Hashem Elsayed Fathi Abd_Allah Altaf Ahmad |
| author_facet | Shruti Singh Maheshwar Prasad Sharma Abdulaziz A. Alqarawi Abeer Hashem Elsayed Fathi Abd_Allah Altaf Ahmad |
| author_sort | Shruti Singh |
| collection | DOAJ |
| description | Isoleucine is one of the branched chain amino acids that plays a major role in the energy metabolism of human beings and animals. However, detailed investigation of specific receptors for isoleucine has not been carried out because of the non-availability of a tool that can monitor the metabolic flux of this amino acid in live cells. This study presents a novel genetically-encoded nanosensor for real-time monitoring of isoleucine in living cells. This nanosensor was developed by sandwiching a periplasmic binding protein (LivJ) of <i>E. coli</i> between a fluorescent protein pair, ECFP (Enhanced Cyan Fluorescent Protein), and Venus. The sensor, named GEII (Genetically Encoded Isoleucine Indicator), was pH stable, isoleucine-specific, and had a binding affinity (K<sub>d</sub>) of 63 ± 6 μM. The GEII successfully performed real-time monitoring of isoleucine in bacterial and yeast cells, thereby, establishing its bio-compatibility in monitoring isoleucine in living cells. As a further enhancement, in silico random mutagenesis was carried out to identify a set of viable mutations, which were subsequently experimentally verified to create a library of affinity mutants with a significantly expanded operating range (96 nM−1493 μM). In addition to its applicability in understanding the underlying functions of receptors of isoleucine in metabolic regulation, the GEII can also be used for metabolic engineering of bacteria for enhanced production of isoleucine in animal feed industries. |
| first_indexed | 2024-04-14T03:28:06Z |
| format | Article |
| id | doaj.art-595e806639e8417999c7606e5989c4de |
| institution | Directory Open Access Journal |
| issn | 1424-8220 |
| language | English |
| last_indexed | 2024-04-14T03:28:06Z |
| publishDate | 2019-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Sensors |
| spelling | doaj.art-595e806639e8417999c7606e5989c4de2022-12-22T02:15:05ZengMDPI AGSensors1424-82202019-12-0120114610.3390/s20010146s20010146Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded NanosensorShruti Singh0Maheshwar Prasad Sharma1Abdulaziz A. Alqarawi2Abeer Hashem3Elsayed Fathi Abd_Allah4Altaf Ahmad5Department of Botany, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi 110062, IndiaDepartment of Botany, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi 110062, IndiaPlant Production Department, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi ArabiaBotany and Microbiology Department, College of Science, King Saud University, P.O. Box. 2460, Riyadh 11451, Saudi ArabiaPlant Production Department, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi ArabiaDepartment of Botany, Aligarh Muslim University, Aligarh 202002, IndiaIsoleucine is one of the branched chain amino acids that plays a major role in the energy metabolism of human beings and animals. However, detailed investigation of specific receptors for isoleucine has not been carried out because of the non-availability of a tool that can monitor the metabolic flux of this amino acid in live cells. This study presents a novel genetically-encoded nanosensor for real-time monitoring of isoleucine in living cells. This nanosensor was developed by sandwiching a periplasmic binding protein (LivJ) of <i>E. coli</i> between a fluorescent protein pair, ECFP (Enhanced Cyan Fluorescent Protein), and Venus. The sensor, named GEII (Genetically Encoded Isoleucine Indicator), was pH stable, isoleucine-specific, and had a binding affinity (K<sub>d</sub>) of 63 ± 6 μM. The GEII successfully performed real-time monitoring of isoleucine in bacterial and yeast cells, thereby, establishing its bio-compatibility in monitoring isoleucine in living cells. As a further enhancement, in silico random mutagenesis was carried out to identify a set of viable mutations, which were subsequently experimentally verified to create a library of affinity mutants with a significantly expanded operating range (96 nM−1493 μM). In addition to its applicability in understanding the underlying functions of receptors of isoleucine in metabolic regulation, the GEII can also be used for metabolic engineering of bacteria for enhanced production of isoleucine in animal feed industries.https://www.mdpi.com/1424-8220/20/1/146isoleucineförster/fluorescence resonance energy transfernanosensorlivjperiplasmic binding protein |
| spellingShingle | Shruti Singh Maheshwar Prasad Sharma Abdulaziz A. Alqarawi Abeer Hashem Elsayed Fathi Abd_Allah Altaf Ahmad Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor Sensors isoleucine förster/fluorescence resonance energy transfer nanosensor livj periplasmic binding protein |
| title | Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor |
| title_full | Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor |
| title_fullStr | Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor |
| title_full_unstemmed | Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor |
| title_short | Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor |
| title_sort | real time optical detection of isoleucine in living cells through a genetically encoded nanosensor |
| topic | isoleucine förster/fluorescence resonance energy transfer nanosensor livj periplasmic binding protein |
| url | https://www.mdpi.com/1424-8220/20/1/146 |
| work_keys_str_mv | AT shrutisingh realtimeopticaldetectionofisoleucineinlivingcellsthroughageneticallyencodednanosensor AT maheshwarprasadsharma realtimeopticaldetectionofisoleucineinlivingcellsthroughageneticallyencodednanosensor AT abdulazizaalqarawi realtimeopticaldetectionofisoleucineinlivingcellsthroughageneticallyencodednanosensor AT abeerhashem realtimeopticaldetectionofisoleucineinlivingcellsthroughageneticallyencodednanosensor AT elsayedfathiabdallah realtimeopticaldetectionofisoleucineinlivingcellsthroughageneticallyencodednanosensor AT altafahmad realtimeopticaldetectionofisoleucineinlivingcellsthroughageneticallyencodednanosensor |