Label-free tracking and mass measurement of single protein complexes on lipid bilayers
<p>Protein-protein interactions are essential for biological processes, but can display a level of polydispersity that is difficult to study with bulk measurements. Recently, mass photometry, a label-free single molecule imaging technique, has been used to quantify protein-protein interactions...
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
2022
|
| Subjects: |
| _version_ | 1797108436455915520 |
|---|---|
| author | Foley, EDB |
| author2 | Kukura, P |
| author_facet | Kukura, P Foley, EDB |
| author_sort | Foley, EDB |
| collection | OXFORD |
| description | <p>Protein-protein interactions are essential for biological processes, but can display a level of polydispersity that is difficult to study with bulk measurements. Recently, mass photometry, a label-free single molecule imaging technique, has been used to quantify protein-protein interactions by measuring the mass of single proteins. So far, however, measurements are limited to solution-based systems and nM concentrations. Here, we aim to expand the applications of mass photometry to proteins on lipid bilayers, and to increase the concentration range of solution-based measurements.</p>
<p>We implement a surface passivation protocol that reduces non-specific binding by up to four orders of magnitude, and apply further surface function- alisation strategies aimed at accessing measurements at μM concentrations. Next, we develop a mass photometry-based method for tracking individual protein complexes on lipid bilayers, thereby demonstrating quantification of oligomer-dependent kinetics and mobilities. We then extend this method to interactions of the HIV-1 envelope glycoprotein (Env) with broadly neutral- ising antibodies. Using our assay, we resolve polydisperse anitbody-induced cross-linking and quantify the corresponding binding affinities. We show that lipid bilayers give access to mass photometry measurements at μM protein concentrations, and demonstrate specific pull-down of target proteins from complex mixtures. Finally, we discuss aspects of the data analysis for these assays with a specific focus on systematic errors in the diffusion and mass measurements, and outline strategies for their correction.</p> |
| first_indexed | 2024-03-07T07:29:12Z |
| format | Thesis |
| id | oxford-uuid:a0c3b677-2393-46a4-99ac-7662276c88a7 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:29:12Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | oxford-uuid:a0c3b677-2393-46a4-99ac-7662276c88a72022-12-15T13:05:12ZLabel-free tracking and mass measurement of single protein complexes on lipid bilayersThesishttp://purl.org/coar/resource_type/c_db06uuid:a0c3b677-2393-46a4-99ac-7662276c88a7MicroscopyHIV antibodiesBilayer lipid membranesBiophysicsEnglishHyrax Deposit2022Foley, EDBKukura, PStruwe, WBenesch, J<p>Protein-protein interactions are essential for biological processes, but can display a level of polydispersity that is difficult to study with bulk measurements. Recently, mass photometry, a label-free single molecule imaging technique, has been used to quantify protein-protein interactions by measuring the mass of single proteins. So far, however, measurements are limited to solution-based systems and nM concentrations. Here, we aim to expand the applications of mass photometry to proteins on lipid bilayers, and to increase the concentration range of solution-based measurements.</p> <p>We implement a surface passivation protocol that reduces non-specific binding by up to four orders of magnitude, and apply further surface function- alisation strategies aimed at accessing measurements at μM concentrations. Next, we develop a mass photometry-based method for tracking individual protein complexes on lipid bilayers, thereby demonstrating quantification of oligomer-dependent kinetics and mobilities. We then extend this method to interactions of the HIV-1 envelope glycoprotein (Env) with broadly neutral- ising antibodies. Using our assay, we resolve polydisperse anitbody-induced cross-linking and quantify the corresponding binding affinities. We show that lipid bilayers give access to mass photometry measurements at μM protein concentrations, and demonstrate specific pull-down of target proteins from complex mixtures. Finally, we discuss aspects of the data analysis for these assays with a specific focus on systematic errors in the diffusion and mass measurements, and outline strategies for their correction.</p> |
| spellingShingle | Microscopy HIV antibodies Bilayer lipid membranes Biophysics Foley, EDB Label-free tracking and mass measurement of single protein complexes on lipid bilayers |
| title | Label-free tracking and mass measurement of single protein complexes on lipid bilayers |
| title_full | Label-free tracking and mass measurement of single protein complexes on lipid bilayers |
| title_fullStr | Label-free tracking and mass measurement of single protein complexes on lipid bilayers |
| title_full_unstemmed | Label-free tracking and mass measurement of single protein complexes on lipid bilayers |
| title_short | Label-free tracking and mass measurement of single protein complexes on lipid bilayers |
| title_sort | label free tracking and mass measurement of single protein complexes on lipid bilayers |
| topic | Microscopy HIV antibodies Bilayer lipid membranes Biophysics |
| work_keys_str_mv | AT foleyedb labelfreetrackingandmassmeasurementofsingleproteincomplexesonlipidbilayers |