FRET-studies of EF-hand complementation and the effect of conjugated fluorescent proteins
(2021) KFKM05 20211Biophysical Chemistry
- Abstract
- In this work spectroscopic methods have been used to determine the effect of fluorophores on protein affinity. Fluorophores are used in many different contexts to mark proteins, genes or antibodies. By marking a construct with a fluorophore, it is possible to follow the localization and function in a biological system, but it is not often investigated how the bound fluorophore could affect the marked construct. To gain some understanding of how marking a protein with a fluorophore might affect the protein properties, the small Ca2+ binding protein calbindin was used. The protein consists of two parts, called EF-hands, with great affinity for each other. The two EF-hands are split up with one fluorophore covalently bound to each EF-hand.... (More)
- In this work spectroscopic methods have been used to determine the effect of fluorophores on protein affinity. Fluorophores are used in many different contexts to mark proteins, genes or antibodies. By marking a construct with a fluorophore, it is possible to follow the localization and function in a biological system, but it is not often investigated how the bound fluorophore could affect the marked construct. To gain some understanding of how marking a protein with a fluorophore might affect the protein properties, the small Ca2+ binding protein calbindin was used. The protein consists of two parts, called EF-hands, with great affinity for each other. The two EF-hands are split up with one fluorophore covalently bound to each EF-hand. There are two different fluorophores which can be positioned on either of the two EF-hands which results in four different constructs and two construct pairs. The two fluorophores have a convenient spectral overlap which allows for excitation energy to travel within the molecules from one fluorophore (donor) to the other fluorophore (acceptor) when they are close enough to each other. When the EF-hands are bound to each other the fluorophores are within range for the excitation energy to travel from donor to acceptor. By measuring the fluorescence decrease from the donor fluorophore when increasing the concentration of the acceptor fluorophore, it is possible to determine equilibrium dissociation constants (KD) of the system. This method can be used at lower protein concentrations on the nM scale, compared to isothermal calorimetry titrations where protein concentrations on the µM scale is needed, whereas SPR is performed on a surface which introduces other artifacts. Using low protein concentrations is convenient since the EF-hands affinity is high and measures very low KD values. From this work, KD values fitted to the experimental data for protein concentrations varying from 3-40 nM were in the range of 0.2-2 nM, whereas the KD values obtained for 80 nM titrations were around 30 nM. One of the construct pairs also had a relatively larger decrease in fluorescence compared to the other construct pair, indicating that the positioning of the fluorophores might affect the distance of the fluorophores. (Less)
- Popular Abstract
- There are many diseases taking countless lives every day, and for many of these diseases we do not know exactly what is happening and why we get ill. The diseases are a result of biological functions going off the rails and malfunctioning mysteriously. By being able to follow a certain biological process without interfering with it, many of the unknown mysteries can be unrolled. But how do we know if we are interfering with a process when we don’t know how it functions to begin with?
I have been working with a protein which can be found in our bodies called calbindin. Proteins are long chain-like structures which can be folded in different ways depending on the elements of the chain, and
they are responsible for all our body functions.... (More) - There are many diseases taking countless lives every day, and for many of these diseases we do not know exactly what is happening and why we get ill. The diseases are a result of biological functions going off the rails and malfunctioning mysteriously. By being able to follow a certain biological process without interfering with it, many of the unknown mysteries can be unrolled. But how do we know if we are interfering with a process when we don’t know how it functions to begin with?
I have been working with a protein which can be found in our bodies called calbindin. Proteins are long chain-like structures which can be folded in different ways depending on the elements of the chain, and
they are responsible for all our body functions. This specific protein can bind Calcium ions, which are metal ions very important for our body to function properly. There are two parts of the protein which each
can bind one calcium ion. The most interesting property of calbindin is, however, that these two parts very strongly want to be together. If they are split up, they will find each other and bind together strongly. This is a characteristic which is very interesting and could be used in many different contexts, that is, if we can follow their binding.
Calbindin is a quite small protein with a short chain, and to follow its functions, much larger proteins, with more than a 5x longer chain are attached to it. These larger proteins have an
ability to emit specific light which makes it possible to follow them. The calbindin protein is split up between its two parts and two different light emitting proteins are added, one for each part
of calbindin. By doing this we can determine, depending on the light emitted from the larger proteins, if the parts of calbindin are bound together or alone. The question here is whether the larger,
light emitting proteins will be in the way when the two parts of calbindin tries to bind together.
My results are that even with much larger light emitting proteins added to the small calbindin protein, the two parts of calbindin can bind together very well. This indicates that the larger proteins are not that much in the way. It is, however, difficult to know how well the calbindin parts would bind together without the larger light emitting proteins, since the other methods to follow the binding have their own sources of error. A conclusion is therefore that, in this case, the larger light emitting proteins do not seem to interfere much with the function of the protein. This information can be used as an assurance when using light emitting proteins to follow biological processes, however, one should not attach new structures to follow a biological process without paying attention to the effect it might have. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9060809
- author
- Håkansson, Cecilia LU
- supervisor
-
- Sara Linse LU
- Thom Leiding LU
- organization
- course
- KFKM05 20211
- year
- 2021
- type
- H3 - Professional qualifications (4 Years - )
- subject
- keywords
- Biophysical chemistry, FRET, EF-hand, complementation, S100G, Protein affinity, Fluorophores
- language
- English
- id
- 9060809
- date added to LUP
- 2021-07-05 15:01:36
- date last changed
- 2021-07-05 15:01:36
@misc{9060809,
abstract = {{In this work spectroscopic methods have been used to determine the effect of fluorophores on protein affinity. Fluorophores are used in many different contexts to mark proteins, genes or antibodies. By marking a construct with a fluorophore, it is possible to follow the localization and function in a biological system, but it is not often investigated how the bound fluorophore could affect the marked construct. To gain some understanding of how marking a protein with a fluorophore might affect the protein properties, the small Ca2+ binding protein calbindin was used. The protein consists of two parts, called EF-hands, with great affinity for each other. The two EF-hands are split up with one fluorophore covalently bound to each EF-hand. There are two different fluorophores which can be positioned on either of the two EF-hands which results in four different constructs and two construct pairs. The two fluorophores have a convenient spectral overlap which allows for excitation energy to travel within the molecules from one fluorophore (donor) to the other fluorophore (acceptor) when they are close enough to each other. When the EF-hands are bound to each other the fluorophores are within range for the excitation energy to travel from donor to acceptor. By measuring the fluorescence decrease from the donor fluorophore when increasing the concentration of the acceptor fluorophore, it is possible to determine equilibrium dissociation constants (KD) of the system. This method can be used at lower protein concentrations on the nM scale, compared to isothermal calorimetry titrations where protein concentrations on the µM scale is needed, whereas SPR is performed on a surface which introduces other artifacts. Using low protein concentrations is convenient since the EF-hands affinity is high and measures very low KD values. From this work, KD values fitted to the experimental data for protein concentrations varying from 3-40 nM were in the range of 0.2-2 nM, whereas the KD values obtained for 80 nM titrations were around 30 nM. One of the construct pairs also had a relatively larger decrease in fluorescence compared to the other construct pair, indicating that the positioning of the fluorophores might affect the distance of the fluorophores.}},
author = {{Håkansson, Cecilia}},
language = {{eng}},
note = {{Student Paper}},
title = {{FRET-studies of EF-hand complementation and the effect of conjugated fluorescent proteins}},
year = {{2021}},
}