Expression and Purification of Channel Proteins : Aiming at structural and functional understanding of TRPAs and Aquaporins
(2024)- Abstract
- Every living cell is surrounded by a cell membrane that is made up of amphipathic
phospholipids, separating the aqueous solutions inside and outside the cell with a
hydrophobic barrier. This compartmentalization is a prerequisite for life, but so are
the many molecules that are floating in the membrane, altering its properties and
connecting the inside with the outside. An important group are the transport
proteins, that open ways for molecules and ions, that would normally be too large,
too charged, or too polar to pass the membrane. The transport proteins are either
active transporters – like pumps – or passive transporters – like channels. In this
thesis, I put the spotlight on two types of channel... (More) - Every living cell is surrounded by a cell membrane that is made up of amphipathic
phospholipids, separating the aqueous solutions inside and outside the cell with a
hydrophobic barrier. This compartmentalization is a prerequisite for life, but so are
the many molecules that are floating in the membrane, altering its properties and
connecting the inside with the outside. An important group are the transport
proteins, that open ways for molecules and ions, that would normally be too large,
too charged, or too polar to pass the membrane. The transport proteins are either
active transporters – like pumps – or passive transporters – like channels. In this
thesis, I put the spotlight on two types of channel proteins: Transient Receptor
Potential ion channels, that let ions pass when activated by temperature or pungent
chemicals, and Aquaporins (AQP), that are mainly responsible for letting water
cross the membrane.
In my work, I have made an effort to study the structure of one TRP member in
particular, known as TRPA1 from pine weevil (Hylobius abietis). I had to find the
best possible conditions to solubilize the protein in detergents, and I have also
investigated other tools such as nanodiscs to keep the protein stable in solution. One
major hurdle has always been the low yields, and it was therefore that a GFP-tag
(Green fluorescent Protein) was added to the protein construct, to facilitate the
tracking of the protein and evaluation of purification methods. Coupled with flow
cytometry, a method for measuring fluorescence and scattering of individual cells,
this proved very useful in designing an expression and purification protocol.
The purified protein was used for Cryo-EM (Electron Microscopy), but the
protein was difficult to freeze on grids with a good homogeneous spread of
individual particles. The use of SRCD (Synchrotron Radiation Circular Dichroism)
proved more successful, and confirmed the secondary structure of the protein, and
gave information on the temperature stability of the protein, with and without
agonists and calcium ions. The rapid evolution of machine learning in the field of
bioinformatics has been of great aid to me, and I have used AlphaFold to predict
several TRPA structures, not just of TRPA1.
I also studied two aquaporins, and their interactions with the FERM-domain of
Ezrin. I used Microscale thermophoresis to determine the dissociation constant
(KD), and found some weak interactions, that may regulate aquaporin trafficking.
Channel proteins are complicated membrane proteins that are hard to express and
purify, but with the help of GFP and various evaluation methods, a lot has been
learned about their structure and function. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/61191c0a-872c-4347-9287-3823093991af
- author
- Werin, Balder LU
- supervisor
- opponent
-
- Professor Drew, David, Stockholms universitet
- organization
- publishing date
- 2024-05-08
- type
- Thesis
- publication status
- published
- subject
- keywords
- TRPA1, Aquaporins, Channel proteins, Pine Weevil, Cryo-EM, Flow cytometry, GFP
- pages
- 157 pages
- publisher
- Lund University
- defense location
- KC:A
- defense date
- 2024-06-03 09:00:00
- ISBN
- 978-91-8096-053-3
- 978-91-8096-052-6
- language
- English
- LU publication?
- yes
- id
- 61191c0a-872c-4347-9287-3823093991af
- date added to LUP
- 2024-05-02 09:50:29
- date last changed
- 2024-06-13 15:03:11
@phdthesis{61191c0a-872c-4347-9287-3823093991af,
abstract = {{Every living cell is surrounded by a cell membrane that is made up of amphipathic<br/>phospholipids, separating the aqueous solutions inside and outside the cell with a<br/>hydrophobic barrier. This compartmentalization is a prerequisite for life, but so are<br/>the many molecules that are floating in the membrane, altering its properties and<br/>connecting the inside with the outside. An important group are the transport<br/>proteins, that open ways for molecules and ions, that would normally be too large,<br/>too charged, or too polar to pass the membrane. The transport proteins are either<br/>active transporters – like pumps – or passive transporters – like channels. In this<br/>thesis, I put the spotlight on two types of channel proteins: Transient Receptor<br/>Potential ion channels, that let ions pass when activated by temperature or pungent<br/>chemicals, and Aquaporins (AQP), that are mainly responsible for letting water<br/>cross the membrane.<br/>In my work, I have made an effort to study the structure of one TRP member in<br/>particular, known as TRPA1 from pine weevil (Hylobius abietis). I had to find the<br/>best possible conditions to solubilize the protein in detergents, and I have also<br/>investigated other tools such as nanodiscs to keep the protein stable in solution. One<br/>major hurdle has always been the low yields, and it was therefore that a GFP-tag<br/>(Green fluorescent Protein) was added to the protein construct, to facilitate the<br/>tracking of the protein and evaluation of purification methods. Coupled with flow<br/>cytometry, a method for measuring fluorescence and scattering of individual cells,<br/>this proved very useful in designing an expression and purification protocol.<br/>The purified protein was used for Cryo-EM (Electron Microscopy), but the<br/>protein was difficult to freeze on grids with a good homogeneous spread of<br/>individual particles. The use of SRCD (Synchrotron Radiation Circular Dichroism)<br/>proved more successful, and confirmed the secondary structure of the protein, and<br/>gave information on the temperature stability of the protein, with and without<br/>agonists and calcium ions. The rapid evolution of machine learning in the field of<br/>bioinformatics has been of great aid to me, and I have used AlphaFold to predict<br/>several TRPA structures, not just of TRPA1.<br/>I also studied two aquaporins, and their interactions with the FERM-domain of<br/>Ezrin. I used Microscale thermophoresis to determine the dissociation constant<br/>(KD), and found some weak interactions, that may regulate aquaporin trafficking.<br/>Channel proteins are complicated membrane proteins that are hard to express and<br/>purify, but with the help of GFP and various evaluation methods, a lot has been<br/>learned about their structure and function.}},
author = {{Werin, Balder}},
isbn = {{978-91-8096-053-3}},
keywords = {{TRPA1; Aquaporins; Channel proteins; Pine Weevil; Cryo-EM; Flow cytometry; GFP}},
language = {{eng}},
month = {{05}},
publisher = {{Lund University}},
school = {{Lund University}},
title = {{Expression and Purification of Channel Proteins : Aiming at structural and functional understanding of TRPAs and Aquaporins}},
url = {{https://lup.lub.lu.se/search/files/182230576/Thesis_LUCRIS.pdf}},
year = {{2024}},
}