Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion
The function of most lipases is controlled by the lid, which undergoes conformational changes at a water–lipid interface to expose the active site, thus activating catalysis. Understanding how lid mutations affect lipases’ function is important for designing improved variants. Li...
| Main Authors: | , , , , , , , |
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| Format: | Article |
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Multidisciplinary Digital Publishing Institute
2023
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| Online Access: | https://hdl.handle.net/1721.1/150487 |
| _version_ | 1811070534009487360 |
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| author | Iversen, Josephine F. Bohr, Søren S.-R. Pinholt, Henrik D. Moses, Matias E. Iversen, Lars Christensen, Sune M. Hatzakis, Nikos S. Zhang, Min |
| author2 | Massachusetts Institute of Technology. Department of Physics |
| author_facet | Massachusetts Institute of Technology. Department of Physics Iversen, Josephine F. Bohr, Søren S.-R. Pinholt, Henrik D. Moses, Matias E. Iversen, Lars Christensen, Sune M. Hatzakis, Nikos S. Zhang, Min |
| author_sort | Iversen, Josephine F. |
| collection | MIT |
| description | The function of most lipases is controlled by the lid, which undergoes conformational changes at a water–lipid interface to expose the active site, thus activating catalysis. Understanding how lid mutations affect lipases’ function is important for designing improved variants. Lipases’ function has been found to correlate with their diffusion on the substrate surface. Here, we used single-particle tracking (SPT), a powerful tool for deciphering enzymes’ diffusional behavior, to study <i>Thermomyces lanuginosus</i> lipase (TLL) variants with different lid structures in a laundry-like application condition. Thousands of parallelized recorded trajectories and hidden Markov modeling (HMM) analysis allowed us to extract three interconverting diffusional states and quantify their abundance, microscopic transition rates, and the energy barriers for sampling them. Combining those findings with ensemble measurements, we determined that the overall activity variation in the application condition is dependent on surface binding and lipase mobility when bound. Specifically, the L4 variant with a TLL-like lid and wild-type (WT) TLL displayed similar ensemble activity, but WT bound stronger to the surface than L4, while L4 had a higher diffusion coefficient and thus activity when bound to the surface. These mechanistic elements can only be de-convoluted by our combined assays. Our findings offer fresh perspectives on the development of the next iteration of enzyme-based detergent. |
| first_indexed | 2024-09-23T08:37:33Z |
| format | Article |
| id | mit-1721.1/150487 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T08:37:33Z |
| publishDate | 2023 |
| publisher | Multidisciplinary Digital Publishing Institute |
| record_format | dspace |
| spelling | mit-1721.1/1504872024-01-10T18:12:18Z Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion Iversen, Josephine F. Bohr, Søren S.-R. Pinholt, Henrik D. Moses, Matias E. Iversen, Lars Christensen, Sune M. Hatzakis, Nikos S. Zhang, Min Massachusetts Institute of Technology. Department of Physics The function of most lipases is controlled by the lid, which undergoes conformational changes at a water–lipid interface to expose the active site, thus activating catalysis. Understanding how lid mutations affect lipases’ function is important for designing improved variants. Lipases’ function has been found to correlate with their diffusion on the substrate surface. Here, we used single-particle tracking (SPT), a powerful tool for deciphering enzymes’ diffusional behavior, to study <i>Thermomyces lanuginosus</i> lipase (TLL) variants with different lid structures in a laundry-like application condition. Thousands of parallelized recorded trajectories and hidden Markov modeling (HMM) analysis allowed us to extract three interconverting diffusional states and quantify their abundance, microscopic transition rates, and the energy barriers for sampling them. Combining those findings with ensemble measurements, we determined that the overall activity variation in the application condition is dependent on surface binding and lipase mobility when bound. Specifically, the L4 variant with a TLL-like lid and wild-type (WT) TLL displayed similar ensemble activity, but WT bound stronger to the surface than L4, while L4 had a higher diffusion coefficient and thus activity when bound to the surface. These mechanistic elements can only be de-convoluted by our combined assays. Our findings offer fresh perspectives on the development of the next iteration of enzyme-based detergent. 2023-04-12T15:41:04Z 2023-04-12T15:41:04Z 2023-03-31 2023-04-12T13:24:08Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/150487 Biomolecules 13 (4): 631 (2023) PUBLISHER_CC http://dx.doi.org/10.3390/biom13040631 Creative Commons Attribution http://creativecommons.org/licenses/by/4.0/ application/pdf Multidisciplinary Digital Publishing Institute Multidisciplinary Digital Publishing Institute |
| spellingShingle | Iversen, Josephine F. Bohr, Søren S.-R. Pinholt, Henrik D. Moses, Matias E. Iversen, Lars Christensen, Sune M. Hatzakis, Nikos S. Zhang, Min Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion |
| title | Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion |
| title_full | Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion |
| title_fullStr | Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion |
| title_full_unstemmed | Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion |
| title_short | Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion |
| title_sort | single particle tracking of thermomyces lanuginosus lipase reveals how mutations in the lid region remodel its diffusion |
| url | https://hdl.handle.net/1721.1/150487 |
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