Engineering adenylate cyclases regulated by near-infrared window light
Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applicat...
| Main Authors: | , , , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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National Academy of Sciences (U.S.)
2015
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| Online Access: | http://hdl.handle.net/1721.1/93769 https://orcid.org/0000-0002-9463-3508 |
| _version_ | 1811084677559091200 |
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| author | Ryu, Min-Hyung Kang, In-Hye Nelson, Mathew D. Jensen, Tricia M. Lyuksyutova, Anna I. Siltberg-Liberles, Jessica Raizen, David M. Gomelsky, Mark |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Ryu, Min-Hyung Kang, In-Hye Nelson, Mathew D. Jensen, Tricia M. Lyuksyutova, Anna I. Siltberg-Liberles, Jessica Raizen, David M. Gomelsky, Mark |
| author_sort | Ryu, Min-Hyung |
| collection | MIT |
| description | Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochrome-based optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset. |
| first_indexed | 2024-09-23T12:55:19Z |
| format | Article |
| id | mit-1721.1/93769 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T12:55:19Z |
| publishDate | 2015 |
| publisher | National Academy of Sciences (U.S.) |
| record_format | dspace |
| spelling | mit-1721.1/937692022-09-28T10:56:17Z Engineering adenylate cyclases regulated by near-infrared window light Ryu, Min-Hyung Kang, In-Hye Nelson, Mathew D. Jensen, Tricia M. Lyuksyutova, Anna I. Siltberg-Liberles, Jessica Raizen, David M. Gomelsky, Mark Massachusetts Institute of Technology. Department of Biological Engineering Ryu, Min-Hyung Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochrome-based optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset. 2015-02-05T15:56:32Z 2015-02-05T15:56:32Z 2014-06 2014-01 Article http://purl.org/eprint/type/JournalArticle 0027-8424 1091-6490 http://hdl.handle.net/1721.1/93769 Ryu, Min-Hyung, In-Hye Kang, Mathew D. Nelson, Tricia M. Jensen, Anna I. Lyuksyutova, Jessica Siltberg-Liberles, David M. Raizen, and Mark Gomelsky. “Engineering Adenylate Cyclases Regulated by Near-Infrared Window Light.” Proceedings of the National Academy of Sciences 111, no. 28 (June 30, 2014): 10167–10172. https://orcid.org/0000-0002-9463-3508 en_US http://dx.doi.org/10.1073/pnas.1324301111 Proceedings of the National Academy of Sciences of the United States of America Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf National Academy of Sciences (U.S.) National Academy of Sciences (U.S.) |
| spellingShingle | Ryu, Min-Hyung Kang, In-Hye Nelson, Mathew D. Jensen, Tricia M. Lyuksyutova, Anna I. Siltberg-Liberles, Jessica Raizen, David M. Gomelsky, Mark Engineering adenylate cyclases regulated by near-infrared window light |
| title | Engineering adenylate cyclases regulated by near-infrared window light |
| title_full | Engineering adenylate cyclases regulated by near-infrared window light |
| title_fullStr | Engineering adenylate cyclases regulated by near-infrared window light |
| title_full_unstemmed | Engineering adenylate cyclases regulated by near-infrared window light |
| title_short | Engineering adenylate cyclases regulated by near-infrared window light |
| title_sort | engineering adenylate cyclases regulated by near infrared window light |
| url | http://hdl.handle.net/1721.1/93769 https://orcid.org/0000-0002-9463-3508 |
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