Interferometric scattering microscopy: from bacterial dynamics to molecular assembly
<p>We explore the capabilities of interferometric scattering microscopy (iSCAT) by investigating a series of biophysical systems that span a range of particle sizes, down to the single particle level. </p> <p>The work presented starts with single particle tracking of 20 nm gold n...
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| Format: | Thesis |
| Language: | English |
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2019
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| _version_ | 1797109034096001024 |
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| author | Fineberg, A |
| author2 | Kukura, P |
| author_facet | Kukura, P Fineberg, A |
| author_sort | Fineberg, A |
| collection | OXFORD |
| description | <p>We explore the capabilities of interferometric scattering microscopy (iSCAT) by investigating a series of biophysical systems that span a range of particle sizes, down to the single particle level. </p>
<p>The work presented starts with single particle tracking of 20 nm gold nanoparticles bound to single myosin 5a molecules. We identify temporally and spatially constrained transient states of the different domains of myosin 5a, shedding light on the structural transitions that the molecule undergoes. The work demonstrates iSCAT's ability to reveal global conformational changes and relative motions of different domains in a single protein.</p>
<p>Next, we make the transition to explore iSCAT's capability for label-free studies of complex biophysical systems. By minimising iSCAT's phototoxicity, we visualise bacterial protein filaments extending from the bacterial cell body. This allows us to understand the roles of different proteins which are employed under varying friction environments to maintain pili function.</p>
<p>By further improving iSCAT's ability to measure smaller scattering signals, we next monitor the assembly kinetics of a MDa-sized gold coordinated protein cage, label-free, in solution. The work demonstrates iSCAT's suitability to determine kinetic information from biological systems, as a dynamic approach complementary to cryoEM.</p>
<p>Lastly, we present an experimental assay to measure the dissociation constant of a simple interaction between two 55 kDa proteins, highlighting the potential for iSCAT to become a universal analytical tool for quantifying protein-protein interactions.</p> |
| first_indexed | 2024-03-07T07:36:24Z |
| format | Thesis |
| id | oxford-uuid:65ada108-6d96-467a-a118-9e3df857d856 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:36:24Z |
| publishDate | 2019 |
| record_format | dspace |
| spelling | oxford-uuid:65ada108-6d96-467a-a118-9e3df857d8562023-03-16T10:40:52ZInterferometric scattering microscopy: from bacterial dynamics to molecular assemblyThesishttp://purl.org/coar/resource_type/c_db06uuid:65ada108-6d96-467a-a118-9e3df857d856BiophysicsMicroscopyLight--ScatteringEnglishORA Deposit2019Fineberg, AKukura, P<p>We explore the capabilities of interferometric scattering microscopy (iSCAT) by investigating a series of biophysical systems that span a range of particle sizes, down to the single particle level. </p> <p>The work presented starts with single particle tracking of 20 nm gold nanoparticles bound to single myosin 5a molecules. We identify temporally and spatially constrained transient states of the different domains of myosin 5a, shedding light on the structural transitions that the molecule undergoes. The work demonstrates iSCAT's ability to reveal global conformational changes and relative motions of different domains in a single protein.</p> <p>Next, we make the transition to explore iSCAT's capability for label-free studies of complex biophysical systems. By minimising iSCAT's phototoxicity, we visualise bacterial protein filaments extending from the bacterial cell body. This allows us to understand the roles of different proteins which are employed under varying friction environments to maintain pili function.</p> <p>By further improving iSCAT's ability to measure smaller scattering signals, we next monitor the assembly kinetics of a MDa-sized gold coordinated protein cage, label-free, in solution. The work demonstrates iSCAT's suitability to determine kinetic information from biological systems, as a dynamic approach complementary to cryoEM.</p> <p>Lastly, we present an experimental assay to measure the dissociation constant of a simple interaction between two 55 kDa proteins, highlighting the potential for iSCAT to become a universal analytical tool for quantifying protein-protein interactions.</p> |
| spellingShingle | Biophysics Microscopy Light--Scattering Fineberg, A Interferometric scattering microscopy: from bacterial dynamics to molecular assembly |
| title | Interferometric scattering microscopy: from bacterial dynamics to molecular assembly |
| title_full | Interferometric scattering microscopy: from bacterial dynamics to molecular assembly |
| title_fullStr | Interferometric scattering microscopy: from bacterial dynamics to molecular assembly |
| title_full_unstemmed | Interferometric scattering microscopy: from bacterial dynamics to molecular assembly |
| title_short | Interferometric scattering microscopy: from bacterial dynamics to molecular assembly |
| title_sort | interferometric scattering microscopy from bacterial dynamics to molecular assembly |
| topic | Biophysics Microscopy Light--Scattering |
| work_keys_str_mv | AT fineberga interferometricscatteringmicroscopyfrombacterialdynamicstomolecularassembly |