Temperature-dependent conformational change of two de novo proteins : Expression, purification and characterisation of two de novo proteins predicted to undergo conformational change at elevated temperatures
(2024) KEML10 20241Department of Chemistry
- Abstract
- Conformational changes are essential for the function of many proteins and is determined by the sequence of amino acids and interactions between them. With computational protein design, new de novo proteins with the ability to undergo conformational changes have been designed. Also, proteins that are one amino acid apart and predicted to have two different conformational states, with the ability to change to the other one can be designed. The aims of this work was to express, purify and characterise two de novo computer-generated proteins, that are one mutation apart and could undergo conformational changes between two structures upon heating. Another aim was to mutate sequences for proteins on a trajectory. To succeed with the aims,... (More)
- Conformational changes are essential for the function of many proteins and is determined by the sequence of amino acids and interactions between them. With computational protein design, new de novo proteins with the ability to undergo conformational changes have been designed. Also, proteins that are one amino acid apart and predicted to have two different conformational states, with the ability to change to the other one can be designed. The aims of this work was to express, purify and characterise two de novo computer-generated proteins, that are one mutation apart and could undergo conformational changes between two structures upon heating. Another aim was to mutate sequences for proteins on a trajectory. To succeed with the aims, Golden Gate Assembly was used to introduce the gene of the proteins in a plasmid. Next the protein was expressed and then analysed with mass spectroscopy, Circular Dichroism (CD) spectroscopy, Nuclear Magnetic Resonance (NMR) and crystallography. Mutations were also done. The results showed that the proteins can undergo conformational change upon heating and that the changes are partially reversible. The results also showed that mutations to the plasmid are possible. The conclusions could be drawn that the two proteins have differences in secondary structure, and that conformational changes happen in response to increased temperature. No conclusions about conformation of mutated proteins were drawn. (Less)
- Popular Abstract
- Proteins are important for all life on earth. They help the body to digest food, move and keep the shape of the cells. Many proteins can change their shape, which can give them a function. These changes can for example happen if a specific molecule binds to the protein, if the charge around the protein changes or even if mechanical forces are used, for instance if you put a hand on your arm. Some proteins can even change their shape if the temperature changes. Because the function of a protein can be dependent of the changes of shape, research is done to try and design proteins with specific shape or ability to change shape. With the help of a computer and different machine learning models, proteins that do not exist in nature can be... (More)
- Proteins are important for all life on earth. They help the body to digest food, move and keep the shape of the cells. Many proteins can change their shape, which can give them a function. These changes can for example happen if a specific molecule binds to the protein, if the charge around the protein changes or even if mechanical forces are used, for instance if you put a hand on your arm. Some proteins can even change their shape if the temperature changes. Because the function of a protein can be dependent of the changes of shape, research is done to try and design proteins with specific shape or ability to change shape. With the help of a computer and different machine learning models, proteins that do not exist in nature can be designed. This is what has been done for the two proteins that have been analysed in this work.
All proteins are built in a sequence, from a bank of 20 smaller building blocks called amino acids. The amino acids have unique characteristics. Some like water, some does not, some have a positive charge, some have a negative charge and some of them have a ring structure that provides them with certain characteristics. The proteins in this work have 150 amino acids each, are designed by a computer and change shape when the temperature increases. The sequences of the building blocks are identical, except for one amino acid that has been changed into another. The computer predicted that this small change would have a big impact on the shape of the protein. In this work, this has been analysed.
The small changes in amino acid sequence, called mutations, are a driving force in the evolution of proteins. These have however mostly been used to explain small changes in the proteins, and not big changes like shape. If small mutations in amino acid sequence can alter the shape of proteins was also analysed in this work.
To do this, the amino acid sequence was ordered and put in bacteria who could create the protein. Then the protein was purified and analysed with different methods. From this, it was concluded that the proteins do change shape when the temperature increases, and the analysed shapes are like the predicted shape to some extent. The method used to change the amino acid sequence did also work. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9158025
- author
- Lundberg, Emma LU
- supervisor
- organization
- course
- KEML10 20241
- year
- 2024
- type
- M2 - Bachelor Degree
- subject
- keywords
- biochemistry, computational protein design, conformational change, protein characterisation, protein evolution, protein expression, protein purification
- language
- English
- id
- 9158025
- date added to LUP
- 2024-06-11 11:01:08
- date last changed
- 2024-06-11 11:01:08
@misc{9158025,
abstract = {{Conformational changes are essential for the function of many proteins and is determined by the sequence of amino acids and interactions between them. With computational protein design, new de novo proteins with the ability to undergo conformational changes have been designed. Also, proteins that are one amino acid apart and predicted to have two different conformational states, with the ability to change to the other one can be designed. The aims of this work was to express, purify and characterise two de novo computer-generated proteins, that are one mutation apart and could undergo conformational changes between two structures upon heating. Another aim was to mutate sequences for proteins on a trajectory. To succeed with the aims, Golden Gate Assembly was used to introduce the gene of the proteins in a plasmid. Next the protein was expressed and then analysed with mass spectroscopy, Circular Dichroism (CD) spectroscopy, Nuclear Magnetic Resonance (NMR) and crystallography. Mutations were also done. The results showed that the proteins can undergo conformational change upon heating and that the changes are partially reversible. The results also showed that mutations to the plasmid are possible. The conclusions could be drawn that the two proteins have differences in secondary structure, and that conformational changes happen in response to increased temperature. No conclusions about conformation of mutated proteins were drawn.}},
author = {{Lundberg, Emma}},
language = {{eng}},
note = {{Student Paper}},
title = {{Temperature-dependent conformational change of two de novo proteins : Expression, purification and characterisation of two de novo proteins predicted to undergo conformational change at elevated temperatures}},
year = {{2024}},
}