Development and Application of Solid-State NMR Methods for Investigating Protein Structure and Dynamics
Solid-state nuclear magnetic resonance spectroscopy (SSNMR) is uniquely well-suited among spectroscopic and microscopy techniques for studying the structure and dynamics of biomacromolecules with atomic length-scale resolution. The development and application of advanced SSNMR techniques facilitates...
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| Format: | Thesis |
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Massachusetts Institute of Technology
2022
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| Online Access: | https://hdl.handle.net/1721.1/139165 https://orcid.org/0000-0002-6412-805X |
| _version_ | 1826211638667116544 |
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| author | Gelenter, Martin D. |
| author2 | Hong, Mei |
| author_facet | Hong, Mei Gelenter, Martin D. |
| author_sort | Gelenter, Martin D. |
| collection | MIT |
| description | Solid-state nuclear magnetic resonance spectroscopy (SSNMR) is uniquely well-suited among spectroscopic and microscopy techniques for studying the structure and dynamics of biomacromolecules with atomic length-scale resolution. The development and application of advanced SSNMR techniques facilitates the study of novel systems to understand protein structure and function.
Obtaining long-range distance restraints is imperative for solving high-resolution protein structures via SSNMR. Third-spin-assisted recoupling (TSAR) experiments have been shown to be extremely useful in obtaining such distance restraints, particularly in the structure determination of fibrils. By replacing the continuous-wave spin-lock with a pulsed spin-lock on the low frequency channels, pulsed TSAR (P-TSAR) experiments reduce the radiofrequency duty cycle of these experiments and makes their optimization more straightforward while maintaining their ability to obtain long distance internuclear contacts.
Glucagon is a peptide hormone that is used as a pharmaceutical agent to treat severe hypoglycemia. Unfortunately, it rapidly fibrillizes at pharmaceutically-relevant concentrations and pH and is thus shipped as a dry lyophilized powder and a diluent solution. Utilizing SSNMR we have solved the high-resolution structure of these cross-β fibrils. Glucagon fibrils consist of alternating antiparallel conformers along the hydrogen bonding fibril axis. In the plane perpendicular to the fibril axis, each conformer forms a symmetric homodimer. Mutations at S2, Y13, A19, or T29 to arginine inhibit fibrillization at pharmaceutical concentrations of glucagon and are promising analogues that would have longer shelf-life in solution compared to wild-type glucagon.
The influenza matrix-2 protein (M2) conducts protons across the lipid membrane in the endosome and is essential for viral replication. Less is known about how the M2 protein of the influenza B strain (BM2) functions compared to AM2 and there are currently no antiviral drugs that are FDA approved that target BM2. Combining SSNMR with molecular dynamics simulations showed that the open, active BM2 channel is more hydrated than the closed, inactive channel, water within the open channel is more dynamic, and water in the open channel has greater orientational anisotropy. The orientational anisotropy is associated with a flip in the orientation of water molecules above and below H19 and is associated with the charge state of H19. |
| first_indexed | 2024-09-23T15:09:10Z |
| format | Thesis |
| id | mit-1721.1/139165 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T15:09:10Z |
| publishDate | 2022 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/1391652022-01-15T03:11:47Z Development and Application of Solid-State NMR Methods for Investigating Protein Structure and Dynamics Gelenter, Martin D. Hong, Mei Massachusetts Institute of Technology. Department of Chemistry Solid-state nuclear magnetic resonance spectroscopy (SSNMR) is uniquely well-suited among spectroscopic and microscopy techniques for studying the structure and dynamics of biomacromolecules with atomic length-scale resolution. The development and application of advanced SSNMR techniques facilitates the study of novel systems to understand protein structure and function. Obtaining long-range distance restraints is imperative for solving high-resolution protein structures via SSNMR. Third-spin-assisted recoupling (TSAR) experiments have been shown to be extremely useful in obtaining such distance restraints, particularly in the structure determination of fibrils. By replacing the continuous-wave spin-lock with a pulsed spin-lock on the low frequency channels, pulsed TSAR (P-TSAR) experiments reduce the radiofrequency duty cycle of these experiments and makes their optimization more straightforward while maintaining their ability to obtain long distance internuclear contacts. Glucagon is a peptide hormone that is used as a pharmaceutical agent to treat severe hypoglycemia. Unfortunately, it rapidly fibrillizes at pharmaceutically-relevant concentrations and pH and is thus shipped as a dry lyophilized powder and a diluent solution. Utilizing SSNMR we have solved the high-resolution structure of these cross-β fibrils. Glucagon fibrils consist of alternating antiparallel conformers along the hydrogen bonding fibril axis. In the plane perpendicular to the fibril axis, each conformer forms a symmetric homodimer. Mutations at S2, Y13, A19, or T29 to arginine inhibit fibrillization at pharmaceutical concentrations of glucagon and are promising analogues that would have longer shelf-life in solution compared to wild-type glucagon. The influenza matrix-2 protein (M2) conducts protons across the lipid membrane in the endosome and is essential for viral replication. Less is known about how the M2 protein of the influenza B strain (BM2) functions compared to AM2 and there are currently no antiviral drugs that are FDA approved that target BM2. Combining SSNMR with molecular dynamics simulations showed that the open, active BM2 channel is more hydrated than the closed, inactive channel, water within the open channel is more dynamic, and water in the open channel has greater orientational anisotropy. The orientational anisotropy is associated with a flip in the orientation of water molecules above and below H19 and is associated with the charge state of H19. Ph.D. 2022-01-14T14:54:04Z 2022-01-14T14:54:04Z 2021-06 2021-06-16T17:13:42.192Z Thesis https://hdl.handle.net/1721.1/139165 https://orcid.org/0000-0002-6412-805X In Copyright - Educational Use Permitted Copyright MIT http://rightsstatements.org/page/InC-EDU/1.0/ application/pdf Massachusetts Institute of Technology |
| spellingShingle | Gelenter, Martin D. Development and Application of Solid-State NMR Methods for Investigating Protein Structure and Dynamics |
| title | Development and Application of Solid-State NMR Methods for Investigating Protein Structure and Dynamics |
| title_full | Development and Application of Solid-State NMR Methods for Investigating Protein Structure and Dynamics |
| title_fullStr | Development and Application of Solid-State NMR Methods for Investigating Protein Structure and Dynamics |
| title_full_unstemmed | Development and Application of Solid-State NMR Methods for Investigating Protein Structure and Dynamics |
| title_short | Development and Application of Solid-State NMR Methods for Investigating Protein Structure and Dynamics |
| title_sort | development and application of solid state nmr methods for investigating protein structure and dynamics |
| url | https://hdl.handle.net/1721.1/139165 https://orcid.org/0000-0002-6412-805X |
| work_keys_str_mv | AT gelentermartind developmentandapplicationofsolidstatenmrmethodsforinvestigatingproteinstructureanddynamics |