A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity
<p dir="ltr">NanoLuc (NLuc) <u>luciferase</u> has found extensive application in designing a range of <u>biologica</u><u>l</u><u> assays,</u> including <u>gene expression analysis</u>, protein-protein interaction, and protei...
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| مؤلفون آخرون: | , , , , , |
| منشور في: |
2024
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| الموضوعات: | |
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إضافة وسم
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| _version_ | 1864513542664749056 |
|---|---|
| author | Wesam S. Ahmed (10170053) |
| author2 | Anupriya M. Geethakumari (17052375) Asfia Sultana (19170880) Asma Fatima (17329974) Angelin M. Philip (13138647) S.M. Nasir Uddin (21841808) Kabir H. Biswas (5705864) |
| author2_role | author author author author author author |
| author_facet | Wesam S. Ahmed (10170053) Anupriya M. Geethakumari (17052375) Asfia Sultana (19170880) Asma Fatima (17329974) Angelin M. Philip (13138647) S.M. Nasir Uddin (21841808) Kabir H. Biswas (5705864) |
| author_role | author |
| dc.creator.none.fl_str_mv | Wesam S. Ahmed (10170053) Anupriya M. Geethakumari (17052375) Asfia Sultana (19170880) Asma Fatima (17329974) Angelin M. Philip (13138647) S.M. Nasir Uddin (21841808) Kabir H. Biswas (5705864) |
| dc.date.none.fl_str_mv | 2024-05-11T09:00:00Z |
| dc.identifier.none.fl_str_mv | 10.1016/j.ijbiomac.2024.131864 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/journal_contribution/A_slow_but_steady_nanoLuc_R162A_mutation_results_in_a_decreased_but_stable_nanoLuc_activity/29715296 |
| dc.rights.none.fl_str_mv | CC BY 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biological sciences Biochemistry and cell biology Engineering Biomedical engineering Bioluminescence Luciferase Molecular dynamics simulation NanoLuc |
| dc.title.none.fl_str_mv | A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity |
| dc.type.none.fl_str_mv | Text Journal contribution info:eu-repo/semantics/publishedVersion text contribution to journal |
| description | <p dir="ltr">NanoLuc (NLuc) <u>luciferase</u> has found extensive application in designing a range of <u>biologica</u><u>l</u><u> assays,</u> including <u>gene expression analysis</u>, protein-protein interaction, and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remain elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable <u>bioluminescence </u>activity of NLuc in living cells and in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the <u>absence</u> and presence of the ligand. Further, analysis of trajectories for <u>hydrogen bonds</u> (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in live cell and in vitro assays performed using lysates prepared from cells expressing the proteins and with the furimazine substrate. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity. </p><h3>Significance </h3><p dir="ltr">Bioluminescence has been extensively utilized in developing a variety of biological and biomedical assays. In this regard, engineering of brighter bioluminescent proteins, i.e. luciferases, has played a significant role. This is acutely exemplified by the engineering of the NLuc <u>luciferase</u>, which is small in size and displays much enhanced bioluminescence and<u> thermal stability</u> compared to previously available luciferases. While enhanced bioluminescent activity is desirable in a multitude of biological and biomedical assays, it would also be useful to develop variants of the protein that display a prolonged bioluminescence activity. This is specifically relevant in designing assays that require bioluminescence for extended periods, such as in the case of biosensors designed for monitoring slow enzymatic or <u>cellular signaling</u> reactions, without necessitating multiple rounds of luciferase substrate addition or any specialized reagents that result in increased assay costs. In the current manuscript, we report a mutant NLuc that possesses a stable and prolonged bioluminescence activity, albeit lower than the wild-type NLuc, and envisage a wider application of the mutant NLuc in designing biosensors for monitoring slower biological and biomedical events.</p><h2>Other Information</h2><p dir="ltr">Published in: International Journal of Biological Macromolecules<br>License: <a href="http://creativecommons.org/licenses/by/4.0/" target="_blank">http://creativecommons.org/licenses/by/4.0/</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1016/j.ijbiomac.2024.131864" target="_blank">https://dx.doi.org/10.1016/j.ijbiomac.2024.131864</a></p> |
| eu_rights_str_mv | openAccess |
| id | Manara2_06d1f9930d23580e9d7ce76936d3f8a4 |
| identifier_str_mv | 10.1016/j.ijbiomac.2024.131864 |
| network_acronym_str | Manara2 |
| network_name_str | Manara2 |
| oai_identifier_str | oai:figshare.com:article/29715296 |
| publishDate | 2024 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activityWesam S. Ahmed (10170053)Anupriya M. Geethakumari (17052375)Asfia Sultana (19170880)Asma Fatima (17329974)Angelin M. Philip (13138647)S.M. Nasir Uddin (21841808)Kabir H. Biswas (5705864)Biological sciencesBiochemistry and cell biologyEngineeringBiomedical engineeringBioluminescenceLuciferaseMolecular dynamics simulationNanoLuc<p dir="ltr">NanoLuc (NLuc) <u>luciferase</u> has found extensive application in designing a range of <u>biologica</u><u>l</u><u> assays,</u> including <u>gene expression analysis</u>, protein-protein interaction, and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remain elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable <u>bioluminescence </u>activity of NLuc in living cells and in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the <u>absence</u> and presence of the ligand. Further, analysis of trajectories for <u>hydrogen bonds</u> (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in live cell and in vitro assays performed using lysates prepared from cells expressing the proteins and with the furimazine substrate. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity. </p><h3>Significance </h3><p dir="ltr">Bioluminescence has been extensively utilized in developing a variety of biological and biomedical assays. In this regard, engineering of brighter bioluminescent proteins, i.e. luciferases, has played a significant role. This is acutely exemplified by the engineering of the NLuc <u>luciferase</u>, which is small in size and displays much enhanced bioluminescence and<u> thermal stability</u> compared to previously available luciferases. While enhanced bioluminescent activity is desirable in a multitude of biological and biomedical assays, it would also be useful to develop variants of the protein that display a prolonged bioluminescence activity. This is specifically relevant in designing assays that require bioluminescence for extended periods, such as in the case of biosensors designed for monitoring slow enzymatic or <u>cellular signaling</u> reactions, without necessitating multiple rounds of luciferase substrate addition or any specialized reagents that result in increased assay costs. In the current manuscript, we report a mutant NLuc that possesses a stable and prolonged bioluminescence activity, albeit lower than the wild-type NLuc, and envisage a wider application of the mutant NLuc in designing biosensors for monitoring slower biological and biomedical events.</p><h2>Other Information</h2><p dir="ltr">Published in: International Journal of Biological Macromolecules<br>License: <a href="http://creativecommons.org/licenses/by/4.0/" target="_blank">http://creativecommons.org/licenses/by/4.0/</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1016/j.ijbiomac.2024.131864" target="_blank">https://dx.doi.org/10.1016/j.ijbiomac.2024.131864</a></p>2024-05-11T09:00:00ZTextJournal contributioninfo:eu-repo/semantics/publishedVersiontextcontribution to journal10.1016/j.ijbiomac.2024.131864https://figshare.com/articles/journal_contribution/A_slow_but_steady_nanoLuc_R162A_mutation_results_in_a_decreased_but_stable_nanoLuc_activity/29715296CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/297152962024-05-11T09:00:00Z |
| spellingShingle | A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity Wesam S. Ahmed (10170053) Biological sciences Biochemistry and cell biology Engineering Biomedical engineering Bioluminescence Luciferase Molecular dynamics simulation NanoLuc |
| status_str | publishedVersion |
| title | A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity |
| title_full | A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity |
| title_fullStr | A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity |
| title_full_unstemmed | A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity |
| title_short | A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity |
| title_sort | A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity |
| topic | Biological sciences Biochemistry and cell biology Engineering Biomedical engineering Bioluminescence Luciferase Molecular dynamics simulation NanoLuc |