Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform
Fluorescently labeled, solute-binding proteins that change their fluorescent output in response to ligand binding are frequently used as biosensors for a wide range of applications. We have previously developed a “Computational Identification of Non-disruptive Conjugation sites” (CINC) approach, an...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-01-01
|
| Series: | Sensors |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1424-8220/22/3/948 |
| _version_ | 1827658831083077632 |
|---|---|
| author | Dustin D. Smith Joshua P. King D. Wade Abbott Hans-Joachim Wieden |
| author_facet | Dustin D. Smith Joshua P. King D. Wade Abbott Hans-Joachim Wieden |
| author_sort | Dustin D. Smith |
| collection | DOAJ |
| description | Fluorescently labeled, solute-binding proteins that change their fluorescent output in response to ligand binding are frequently used as biosensors for a wide range of applications. We have previously developed a “Computational Identification of Non-disruptive Conjugation sites” (CINC) approach, an in silico pipeline utilizing molecular dynamics simulations for the rapid design and construction of novel protein–fluorophore conjugate-type biosensors. Here, we report an improved in silico scoring algorithm for use in CINC and its use in the construction of an oligogalacturonide-detecting biosensor set. Using both 4,5-unsaturated and saturated oligogalacturonides, we demonstrate that signal transmission from the ligand-binding pocket of the starting protein scaffold to the CINC-selected reporter positions is effective for multiple different ligands. The utility of an oligogalacturonide-detecting biosensor is shown in Carbohydrate Active Enzyme (CAZyme) activity assays, where the biosensor is used to follow product release upon polygalacturonic acid (PGA) depolymerization in real time. The oligogalacturonide-detecting biosensor set represents a novel enabling tool integral to our rapidly expanding platform for biosensor-based carbohydrate detection, and moving forward, the CINC pipeline will continue to enable the rational design of biomolecular tools to detect additional chemically distinct oligosaccharides and other solutes. |
| first_indexed | 2024-03-09T23:09:10Z |
| format | Article |
| id | doaj.art-c560f8d1f4314f0ca1688dbfca4d995b |
| institution | Directory Open Access Journal |
| issn | 1424-8220 |
| language | English |
| last_indexed | 2024-03-09T23:09:10Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Sensors |
| spelling | doaj.art-c560f8d1f4314f0ca1688dbfca4d995b2023-11-23T17:48:00ZengMDPI AGSensors1424-82202022-01-0122394810.3390/s22030948Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design PlatformDustin D. Smith0Joshua P. King1D. Wade Abbott2Hans-Joachim Wieden3Alberta RNA Research and Training Institute (ARRTI), University of Lethbridge, Lethbridge, AB T1K 3M4, CanadaAlberta RNA Research and Training Institute (ARRTI), University of Lethbridge, Lethbridge, AB T1K 3M4, CanadaDepartment of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4, CanadaAlberta RNA Research and Training Institute (ARRTI), University of Lethbridge, Lethbridge, AB T1K 3M4, CanadaFluorescently labeled, solute-binding proteins that change their fluorescent output in response to ligand binding are frequently used as biosensors for a wide range of applications. We have previously developed a “Computational Identification of Non-disruptive Conjugation sites” (CINC) approach, an in silico pipeline utilizing molecular dynamics simulations for the rapid design and construction of novel protein–fluorophore conjugate-type biosensors. Here, we report an improved in silico scoring algorithm for use in CINC and its use in the construction of an oligogalacturonide-detecting biosensor set. Using both 4,5-unsaturated and saturated oligogalacturonides, we demonstrate that signal transmission from the ligand-binding pocket of the starting protein scaffold to the CINC-selected reporter positions is effective for multiple different ligands. The utility of an oligogalacturonide-detecting biosensor is shown in Carbohydrate Active Enzyme (CAZyme) activity assays, where the biosensor is used to follow product release upon polygalacturonic acid (PGA) depolymerization in real time. The oligogalacturonide-detecting biosensor set represents a novel enabling tool integral to our rapidly expanding platform for biosensor-based carbohydrate detection, and moving forward, the CINC pipeline will continue to enable the rational design of biomolecular tools to detect additional chemically distinct oligosaccharides and other solutes.https://www.mdpi.com/1424-8220/22/3/948computational biosensor designmolecular dynamicsfluorescencerapid kineticscarbohydrate detectionoligogalacturonides |
| spellingShingle | Dustin D. Smith Joshua P. King D. Wade Abbott Hans-Joachim Wieden Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform Sensors computational biosensor design molecular dynamics fluorescence rapid kinetics carbohydrate detection oligogalacturonides |
| title | Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform |
| title_full | Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform |
| title_fullStr | Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform |
| title_full_unstemmed | Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform |
| title_short | Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform |
| title_sort | development of a real time pectic oligosaccharide detecting biosensor using the rapid and flexible computational identification of non disruptive conjugation sites cinc biosensor design platform |
| topic | computational biosensor design molecular dynamics fluorescence rapid kinetics carbohydrate detection oligogalacturonides |
| url | https://www.mdpi.com/1424-8220/22/3/948 |
| work_keys_str_mv | AT dustindsmith developmentofarealtimepecticoligosaccharidedetectingbiosensorusingtherapidandflexiblecomputationalidentificationofnondisruptiveconjugationsitescincbiosensordesignplatform AT joshuapking developmentofarealtimepecticoligosaccharidedetectingbiosensorusingtherapidandflexiblecomputationalidentificationofnondisruptiveconjugationsitescincbiosensordesignplatform AT dwadeabbott developmentofarealtimepecticoligosaccharidedetectingbiosensorusingtherapidandflexiblecomputationalidentificationofnondisruptiveconjugationsitescincbiosensordesignplatform AT hansjoachimwieden developmentofarealtimepecticoligosaccharidedetectingbiosensorusingtherapidandflexiblecomputationalidentificationofnondisruptiveconjugationsitescincbiosensordesignplatform |