Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum
Photosynthetic organisms have evolved a wide range of strategies to circumvent the limitations of Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). A carbon concentrating mechanism (CCM) is a strategy that collectively describes the organism’s method to increase substrate CO2 for Rubisco to...
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| Format: | Thesis-Doctor of Philosophy |
| Language: | English |
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Nanyang Technological University
2023
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| Online Access: | https://hdl.handle.net/10356/164697 |
| _version_ | 1811691257593856000 |
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| author | Ang, Warren Shou Leong |
| author2 | Oliver Mueller-Cajar |
| author_facet | Oliver Mueller-Cajar Ang, Warren Shou Leong |
| author_sort | Ang, Warren Shou Leong |
| collection | NTU |
| description | Photosynthetic organisms have evolved a wide range of strategies to circumvent the limitations of Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). A carbon concentrating mechanism (CCM) is a strategy that collectively describes the organism’s method to increase substrate CO2 for Rubisco to achieve maximum functional capacity. Herein, we characterize part of the CCM of the diatom Phaeodactylum tricornutum through in vitro reconstitution of its pyrenoid. Studies prior to this project resulted in the discovery of an intrinsically disordered pyrenoid scaffold protein comprised of six tandem repeat sequences, Pyrenoid component 1 (PYCO1) which can form a dense phase to specifically recruit P. tricornutum Rubisco (PtRubisco). It was established that these interactions occur through “KWSPR/Q” motifs on PYCO1 with PtRubisco small subunits. In this study, more detailed characterization of these biomolecular condensates were carried out which elucidated several key findings. Rubisco was found to be enriched up to 200-fold in these condensates. Each PYCO1 molecule sequesters up to 3 units of Rubisco holoenzymes in fully-saturated condensates. The mobility of components in these condensates also change according to Rubisco occupancy. Cryo-EM structural analysis confirmed the interaction of the “KWSPR” motif with Rubisco. Additional motifs on the C-terminus of PYCO1 also interact with Rubisco through an alpha helical motif conserved within P. tricornutum. The data suggests that the “KWSPR” stickers must dimerize prior to binding the SSU of Rubisco. Specific aromatic residues on PYCO1 are required for both heterotypic and homotypic phase separation of the system. Additional pulldown experiments on pyrenoid components led to the identification of a novel protein with the accession number Phatr3_J47612. Phatr3_J47612 and its fragments partition into PYCO1 condensates and antagonize the recruitment of PtRubisco. The likely next steps in this project would be directed towards a systematic study of the interplay of several pyrenoidal proteins to develop our understanding of the diatom CCM. |
| first_indexed | 2024-10-01T06:17:01Z |
| format | Thesis-Doctor of Philosophy |
| id | ntu-10356/164697 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T06:17:01Z |
| publishDate | 2023 |
| publisher | Nanyang Technological University |
| record_format | dspace |
| spelling | ntu-10356/1646972023-03-06T07:30:04Z Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum Ang, Warren Shou Leong Oliver Mueller-Cajar School of Biological Sciences cajar@ntu.edu.sg Science::Biological sciences Photosynthetic organisms have evolved a wide range of strategies to circumvent the limitations of Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). A carbon concentrating mechanism (CCM) is a strategy that collectively describes the organism’s method to increase substrate CO2 for Rubisco to achieve maximum functional capacity. Herein, we characterize part of the CCM of the diatom Phaeodactylum tricornutum through in vitro reconstitution of its pyrenoid. Studies prior to this project resulted in the discovery of an intrinsically disordered pyrenoid scaffold protein comprised of six tandem repeat sequences, Pyrenoid component 1 (PYCO1) which can form a dense phase to specifically recruit P. tricornutum Rubisco (PtRubisco). It was established that these interactions occur through “KWSPR/Q” motifs on PYCO1 with PtRubisco small subunits. In this study, more detailed characterization of these biomolecular condensates were carried out which elucidated several key findings. Rubisco was found to be enriched up to 200-fold in these condensates. Each PYCO1 molecule sequesters up to 3 units of Rubisco holoenzymes in fully-saturated condensates. The mobility of components in these condensates also change according to Rubisco occupancy. Cryo-EM structural analysis confirmed the interaction of the “KWSPR” motif with Rubisco. Additional motifs on the C-terminus of PYCO1 also interact with Rubisco through an alpha helical motif conserved within P. tricornutum. The data suggests that the “KWSPR” stickers must dimerize prior to binding the SSU of Rubisco. Specific aromatic residues on PYCO1 are required for both heterotypic and homotypic phase separation of the system. Additional pulldown experiments on pyrenoid components led to the identification of a novel protein with the accession number Phatr3_J47612. Phatr3_J47612 and its fragments partition into PYCO1 condensates and antagonize the recruitment of PtRubisco. The likely next steps in this project would be directed towards a systematic study of the interplay of several pyrenoidal proteins to develop our understanding of the diatom CCM. Doctor of Philosophy 2023-02-13T02:00:12Z 2023-02-13T02:00:12Z 2022 Thesis-Doctor of Philosophy Ang, W. S. L. (2022). Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/164697 https://hdl.handle.net/10356/164697 10.32657/10356/164697 en This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). application/pdf Nanyang Technological University |
| spellingShingle | Science::Biological sciences Ang, Warren Shou Leong Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum |
| title | Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum |
| title_full | Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum |
| title_fullStr | Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum |
| title_full_unstemmed | Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum |
| title_short | Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum |
| title_sort | characterization of a rubisco condensate derived from the red type pyrenoid of phaeodactylum tricornutum |
| topic | Science::Biological sciences |
| url | https://hdl.handle.net/10356/164697 |
| work_keys_str_mv | AT angwarrenshouleong characterizationofarubiscocondensatederivedfromtheredtypepyrenoidofphaeodactylumtricornutum |