C. Protein Analysis Tools Flashcards
1
Q
- Protein ______ tools and techniques enable one to separate proteins and subsequently identify them.
A
analysis
2
Q
- Separation and identification methods include: (4)
A
- SDS-PAGE
- 2D-Gel Electrophoresis
- Isoelectric focusing (IEF)
- Western blot
3
Q
- Separation and identification methods include: (4)
A
- SDS-PAGE
- 2D-Gel Electrophoresis
- Isoelectric focusing (IEF)
- Western blot
4
Q
- Protein purification of methods include _________.
- Insulin is a protein that is produced in a lab for patients who cannot make it themselves. It is
important to use analysis tools to ensure the correct protein (_______) has been made and that it contains no other proteins that could be harmful to people.
A
chromatography
insulin
5
Q
What does SDS-PAGE stand for?
A
- SDS-PAGE stands for Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis.
6
Q
How does SDS-PAGE work? (5)
A
- A solid medium in the form of a gel is submerged in a buffer solution.
- The gel has wells on top in which a protein sample can be transferred using a pipette.
- Because the density of the protein sample is typically similar to that of the buffer solution, the
sample’s density is usually increased by mixing it with a loading dye enabling the sample to be
suspended in the wells of the gel. - The gel enables us to see the migration of the protein across the gel upon applying an electric
current. - The protein migrates from the cathode end (negative) to the anode end (positive) of the gel,
enabling the protein to migrate along the gel in the same direction.
7
Q
How does SDS-PAGE work? (5)
A
- A solid medium in the form of a gel is submerged in a buffer solution.
- The gel has wells on top in which a protein sample can be transferred using a pipette.
- Because the density of the protein sample is typically similar to that of the buffer solution, the
sample’s density is usually increased by mixing it with a loading dye enabling the sample to be
suspended in the wells of the gel. - The gel enables us to see the migration of the protein across the gel upon applying an electric
current. - The protein migrates from the cathode end (negative) to the anode end (positive) of the gel,
enabling the protein to migrate along the gel in the same direction.
8
Q
What is SDS? (3)
A
- SDS (Sodium Dodecyl Sulphate) is an amphipathic detergent with an anionic tail and a lipophilic tail.
- SDS is used to denature and dissociate proteins from each other and confers a negative charge on
the protein, masking the protein’s intrinsic charge. - SDS-treated proteins have similar mass to charge ratios and shapes.
9
Q
What happens during PAGE? (2)
A
- During PAGE (Polyacrylamide Gel Electrophoresis), the rate of protein migration is determined by molecular weight, where all protein migrates from the cathode to the anode.
- PAGE is the support medium, and the gel has pores which are determined by the concentration of the acrylamide used to prepare it. A lower concentration of acrylamide will mean larger pores and vice versa.
10
Q
PAGE:
- In a gel with uniform density, the relative migration distance of protein (Rf) is ______
_______ to the log of its mass. - Performing a gel with proteins of known and unknown molecular masses simultaneously allows one to estimate the masses of the unknown proteins by plotting the relationship between ___ and the log of its mass.
A
Negatively Proportional
Rf
11
Q
What is 2D-Gel Electrophoresis? (3)
A
- The aim of 2D-Gel Electrophoresis to analyse complex protein mixtures from cells, tissues and other biological samples.
- 2D-Gel Electrophoresis is a combination of Isoelectric Focusing (explained on next page) and SDS- PAGE (explained above).
- This is done by separating and identifying proteins in two steps or two dimensions.
12
Q
2D-Gel Electrophoresis
What are the steps involved? (2)
A
Step 1 – Isoelectric focusing (IEF):
Proteins are separated according to their isoelectric points (the pH at which a particular molecule carries no net electrical charge).
Step 2 – SDS-PAGE
Proteins are separated according to their mass.
13
Q
2D-Gel Electrophoresis
What are the steps involved? (2)
A
Step 1 – Isoelectric focusing (IEF):
Proteins are separated according to their isoelectric points (the pH at which a particular molecule carries no net electrical charge).
Step 2 – SDS-PAGE
Proteins are separated according to their mass.
14
Q
What is Isoelectric Focusing (IEF)? (8)
A
- Isoelectric focusing is used for the analysis of complex protein mixtures from cells.
- A solution with proteins of various molecular masses and different charges are separated according
to their isoelectric points, where upon the application of an electric current through the gel matrix,
the protein becomes stationary at the point where its net charge is 0. - A pH gradient is applied onto a gel and an electric current is applied across the gel, making one end
more positive than the other. - Proteins are charged at all pH values besides their isoelectric values (the pH at which a particular
molecule carries no net electric charge). - Proteins migrate along the gel until they reach their isoelectric points, where they remain
stationary. - Positively charged proteins are pulled towards the negative end of the gel and vice versa.
- The pH gradient in the gel is formed by the presence of ampholytes, which are complex mixtures of
synthetic polyamino-polycarboxylic acids. - In Isoelectric focusing, proteins are separated because of their charge rather than their molecular
mass.
15
Q
What is Protein Purification: Chromatography?
A
- Proteins must be purified to ensure that the highest purity of protein is obtained.
- Pure protein does not have additional cell components, or other protein and/or contaminants that can contribute to the efficient functionality of the protein of interest.
- Chromatography is a fundamental component of most, if not all, recombinant protein purification.
- There are many types of chromatography, but the basic principle is the same. A sample containing the desired protein is applied to a solid matrix and allowed to elute (remove an adsorbed substance by washing with a solvent) through a porous plug where the eluate (a solution obtained by elution) can be collected.
- Over time, the sample is applied to the matrix, and various fractions of the sample can be collected, based on the type of chromatography used for the purification.
16
Q
What is Protein Purification: Chromatography?
A
- Proteins must be purified to ensure that the highest purity of protein is obtained.
- Pure protein does not have additional cell components, or other protein and/or contaminants that can contribute to the efficient functionality of the protein of interest.
- Chromatography is a fundamental component of most, if not all, recombinant protein purification.
- There are many types of chromatography, but the basic principle is the same. A sample containing the desired protein is applied to a solid matrix and allowed to elute (remove an adsorbed substance by washing with a solvent) through a porous plug where the eluate (a solution obtained by elution) can be collected.
- Over time, the sample is applied to the matrix, and various fractions of the sample can be collected, based on the type of chromatography used for the purification.
17
Q
There are 3 main types of chromatography, based on the protein that is the basis of the purification: (3)
A
1) Affinity-Based Chromatography
2) Size-Exclusion Chromatography
3) Ion-Exchange Chromatography
18
Q
What is 1) Affinity-Based Chromatography?
A
The solid matrix, which is usually composed of beads, is coated with a molecule which has an affinity for the protein.
For example, the beads maybe coated with an antigen that has a high affinity to the protein, therefore causing the protein to bind to the beads, and not be eluted with the rest of the sample. The protein can then be collected using a relevant solvent that breaks the bond between the antigen and the protein of interest, and the protein fragment can be collected.
19
Q
What is 1) Affinity-Based Chromatography?
A
The solid matrix, which is usually composed of beads, is coated with a molecule which has an affinity for the protein.
For example, the beads maybe coated with an antigen that has a high affinity to the protein, therefore causing the protein to bind to the beads, and not be eluted with the rest of the sample. The protein can then be collected using a relevant solvent that breaks the bond between the antigen and the protein of interest, and the protein fragment can be collected.
20
Q
What is 2) Size-Exclusion Chromatography?
A
The solid matrix traps the protein within pores in the solid matrix. Chromatography beads with specific pore sizes can be purchased to enable entrapment of the protein (of known size) of interest.
21
Q
What is 3) Ion-Exchange Chromatography?
A
Ion-exchange chromatography facilitates binding of the protein of interest to the solid matrix based on opposing charges.
For example, in the image alongside, the solid matrix composed of beads is positively charged and binds the negatively charged protein molecules.
22
Q
Pure Protein:
- In lane 2 in the image below, an _____-______ protein (pure protein) is shown, whereas lane 1 shows the banding pattern for the total protein lysate, prior to purification.
- Once a protein has been purified, there are many ways in which it may be utilised, as shown below.
A
affinity-purified
23
Q
What is the Recombinant DNA (rDNA)?
A
Recombinant DNA (rDNA) is a DNA strand that is formed by 2 or more DNA sequences which are often from different organisms. The resulting Recombinant DNA is put into a host cell where it is expressed into a new protein, called a Recombinant Protein (rProtein).
24
Q
What is the Recombinant DNA (rDNA)?
A
Recombinant DNA (rDNA) is a DNA strand that is formed by 2 or more DNA sequences which are often from different organisms. The resulting Recombinant DNA is put into a host cell where it is expressed into a new protein, called a Recombinant Protein (rProtein).
25
Recombinant DNA (rDNA):
- Proteins are the most abundant molecules of living organisms and they play an important role in structural and ______ organisation of the cell.
- Recombinant technology refers to the recombination of genetic material of one organism with another ___ ______ (in the lab).
- Recombinant material is introduced into a host cell.
- Recombinant proteins (rProtein) result from the ______ of recombinant DNA (rDNA) within
living cells.
- Once rDNA is inserted into bacteria, these bacteria will make protein based on the rDNA being
translated into rProtein. This occurs like normal gene expression, where DNA is transcribed into mRNA, which is then ______ into protein.
functional
in vitro
expression
translated
26
Cloning Expression Cassettes
- Cloning expression cassettes enables protein expression and is illustrated as follows: (5)
1. The gene responsible for the protein is isolated from the DNA of a human cell using restriction enzymes.
2. The same restriction enzymes are used to cut a piece of DNA from a plasmid of a bacterial cell in the example above. This does not necessarily have to be a plasmid from a bacterial cell.
3. The DNA cut from the human cell (the gene of interest) is then joined or ligated to the piece of DNA from the plasmid (in this specific example) obtained from the bacterial cell.
4. This leads to the development of a recombinant plasmid/vector which is then inserted into a bacterial host cell which multiplies with the recombinant plasmid within it.
5. Note that in this example plasmid was used as a vector. There are other vectors that can be used.
27
Cloning Expression Cassettes
- Cloning expression cassettes enables protein expression and is illustrated as follows: (5)
1. The gene responsible for the protein is isolated from the DNA of a human cell using restriction enzymes.
2. The same restriction enzymes are used to cut a piece of DNA from a plasmid of a bacterial cell in the example above. This does not necessarily have to be a plasmid from a bacterial cell.
3. The DNA cut from the human cell (the gene of interest) is then joined or ligated to the piece of DNA from the plasmid (in this specific example) obtained from the bacterial cell.
4. This leads to the development of a recombinant plasmid/vector which is then inserted into a bacterial host cell which multiplies with the recombinant plasmid within it.
5. Note that in this example plasmid was used as a vector. There are other vectors that can be used.
28
Recombinant Protein Expression:
- As mentioned, rProteins involve the introduction of rDNA into a host cell, where the cellular machinery will be utilised to express _______.
- To achieve this, a process called _____ ________ is performed when the host cell is of bacterial origin and is referred to as Cell Transfection when a Eukaryotic cell is used as the host cell.
- Recall that cells have cell membranes which regulate entry and exit of molecules into and out of the cell to protect the cell from _______ molecules which may harm the cell.
- Transformation and Transfection enable the cell membrane to become ______ to allow the foreign molecule (in this case rDNA) to enter into the cell.
- Transformation can be done through Electroporation (where the cell membrane is treated with an electric current to reach sufficient permeability for the introduction of rDNA into the cell) or _______ (by calcium salts and heat shock).
- Transfection can be done through Electroporation (same as above), Chemically (with calcium salts which is an old method, or with Lipids which is a modern method), or through ________.
- The ________ of the protein lead to the protein being harvested adequately.
rProtein
Bacterial Transformation
foreign
porous
chemically
Microinjection
Overexpression
29
What are the advantages or challenges of each?
30
Protein Expression and Purification Summary: (5)
- A gene on interest is isolated.
- The isolated gene of interest is inserted into an expression vector, resulting in a recombinant vector
or recombinant DNA.
- The recombinant vector/recombinant DNA is transferred into a host cell through Transformation (if
the host cell is bacterial) or transfection (if the host cell is Eukaryotic).
- The cell containing the recombinant proteins are then identified and isolated, and those cells are
allowed to grow.
- The protein is then isolated and purified.
31
What are Cloning Vectors?
- Cloning vectors have important features that enable them to play their role as carries of DNA fragments/genes of interest.
32
- A plasmid vector called pBR322 is shown alongside. This is a commonly used plasmid and is used as an example to illustrate the required features of a cloning vector, which are: (2)
1) Origin of replication (the site where DNA replication is initiated).
2) Marker genes for selection and/or screening.
32
- A plasmid vector called pBR322 is shown alongside. This is a commonly used plasmid and is used as an example to illustrate the required features of a cloning vector, which are: (4)
1) Origin of replication (the site where DNA replication is initiated).
2) Marker genes for selection and/or screening.
3) Unique restriction endonuclease (RE) sites (sometimes called restriction sites). Allows inserts to be cloned in specific sites on the plasmid.
4) Promoters for gene expression.
Allows expression of a cloned gene in the vector.
33
2) Marker genes for selection and/or screening.
Selectable Markers:
Only cells containing plasmid forms colonies.
An example of a selectable marker is antibiotic resistance. Cells that are resistant to antibiotics can grow on media containing antibiotic, and this means that only the cell containing the plasmid will form colonies, because if the cells do not contain the plasmid/recombinant vector, they will not have the antibiotic resistance gene and will die in the presence of antibiotic, allowing for selection and identification of the plasmid-containing cells.
34
2) Marker genes for selection and/or screening.
Screenable marker for recombinant molecules:
Allows screening of different phenotypes.
An example of a screenable marker for recombinant molecules are genes which express a colour change in the presence of a substrate, thus allowing screening of the different phenotypes.
34
2) Marker genes for selection and/or screening.
Screenable marker for recombinant molecules:
Allows screening of different phenotypes.
An example of a screenable marker for recombinant molecules are genes which express a colour change in the presence of a substrate, thus allowing screening of the different phenotypes.
35
- Vectors are self-replicating DNA molecules used to transfer foreign DNA between host cells.
- Ideal vectors are small in size with at least one restriction endonuclease site or endonuclease
enzyme. Most cloning vector systems have been highly commercialised and have been used in many research projects, and as such, the average vector has multiple endonuclease sites.
- Examples of vectors include: (3)
1) Plasmids, which are contained in bacteria. They are extrachromosomal molecules that are circular and double-stranded. They are able to replicate independently of the genomic material in the cell.
2) Bacteriophages, which are viruses that infect bacteria. Bacteriophages multiply within the host cell and are released from the host cell as they multiply. If the bacteriophage contains a gene of interest, it too will multiply with the bacteriophages.
3) Cosmids, which are specialised plasmids with DNA sequences called cos sites, to which foreign DNA can be inserted.
36
- Vectors are self-replicating DNA molecules used to transfer foreign DNA between host cells.
- Ideal vectors are small in size with at least one restriction endonuclease site or endonuclease
enzyme. Most cloning vector systems have been highly commercialised and have been used in many research projects, and as such, the average vector has multiple endonuclease sites.
- Examples of vectors include: (3)
1) Plasmids, which are contained in bacteria. They are extrachromosomal molecules that are circular and double-stranded. They are able to replicate independently of the genomic material in the cell.
2) Bacteriophages, which are viruses that infect bacteria. Bacteriophages multiply within the host cell and are released from the host cell as they multiply. If the bacteriophage contains a gene of interest, it too will multiply with the bacteriophages.
3) Cosmids, which are specialised plasmids with DNA sequences called cos sites, to which foreign DNA can be inserted.
37
The Development of Recombinant Insulin: (5)
*cDNA is complementary DNA.
38
Applications of Recombinant Proteins
Medical applications: (7)
- Haematopoietic growth factor (to do with making blood).
- Hormones.
- Interferons (signalling proteins involved in the immune system).
- rProteins.
- Tissue/bone growth factors and clotting factors.
- Biological response modifiers.
- Medical research.
39
Area of treatment:
Example of protein:
40
- Most enzymes are ______.
- Some enzymes are RNA molecules and are called ______. An example of this is Peptidyl
Transferase which catalyses the formation of the protein bond in protein synthesis.
- Enzymes are highly specific catalysts that accelerate the rate of a reaction. They produce a new
product from the substrate that they act on. They enhance the specificity of that _______ chemical reaction.
- Kinases are an example of enzymes.
proteins
ribozymes
metabolic
41
- Enzymes have an active site within the protein to which the substrates (target molecule) of the reaction bind.
- In the case of kinases, the substrates are the target protein and ____, and they would bind to the target site of that enzyme.
- Enzymes reduce the amount of activation energy needed to reach a _____ _____, thereby speeding up the reaction.
- Binding to the enzyme places strain on bonds in the substrate and lowers the energy needed to break and form new bonds and hence form the products.
- In the case of kinases, the products would be a protein with a ______ attached (Protein-P) and ADP.
ATP
Transition State
Phosphate
42
- For kinases:
Substrates Products
Target Protein + ____ ➔ Target Protein-P + ____
ATP
ADP
43
Kinases
- There are over ____ protein kinases in the human genome.
- Protein kinases comprise 2% of human genes.
- They have evolved from a common ancestral gene, and they share sequence ______.
- Protein structural domains are important for their functions.
- ____% of human proteins can be modified by kinase activity.
- Kinases are enzymes that catalyse the __________ (addition of phosphate) of proteins.
- Kinases take a phosphate from Adenosine Triphosphate (ATP) and link it to a protein, thereby
yielding a protein with a phosphate attached and Adenosine Diphosphate (ADP).
- This reaction is unidirectional because of the large amount of energy released when the phosphate
bond in ATP is ______.
500
similarity
30%
phosphorylation
broken
44
What happens when kinases are added to this reaction?
45
- Protein kinases phosphorylates a specific sequence motif. This means that is not just the ______, ______ or ______ that is recognised, but it is the sequence of amino acids on either side of the
target Serine, Threonine or Tyrosine that is recognised by the protein kinase.
- Each kinase recognises its own ______ sequence.
- Some kinases phosphorylate only one protein, whereas others can phosphorylate many proteins.
Thus, kinases activated by different signalling pathways can result in _______ of the same protein.
- Serine, Threonine or Tyrosine
- consensus
- phosphorylation
46
What is Protein Phosphorylation? (4)
- More than one site on a polypeptide chain can be phosphorylated. This means there can be multiple amino acids within a protein that can be phosphorylated.
- Phosphorylation adds two negative charges to a protein. Note the O- -→
- Recall that the structure of a protein depends on the interactions between the variable side chains of its amino acids. Introducing a negative charge onto that polypeptide chain will change how these different variable side chains interact with each other. Other negatively charged amino acids in the vicinity will move away from these phosphorylated amino acids and positively charged amino acids will be attracted.
- Phosphorylation causes a conformational change in the protein and changes its shape, and the shape of a protein determines its function.
47
What is Protein Phosphorylation? (4)
- More than one site on a polypeptide chain can be phosphorylated. This means there can be multiple amino acids within a protein that can be phosphorylated.
- Phosphorylation adds two negative charges to a protein. Note the O- -→
- Recall that the structure of a protein depends on the interactions between the variable side chains of its amino acids. Introducing a negative charge onto that polypeptide chain will change how these different variable side chains interact with each other. Other negatively charged amino acids in the vicinity will move away from these phosphorylated amino acids and positively charged amino acids will be attracted.
- Phosphorylation causes a conformational change in the protein and changes its shape, and the shape of a protein determines its function.
48
- Thus, adding the phosphate group to a protein could change its: (3)
- Activity (increase or decrease).
- Interactions with other proteins, because if its shape changes and it is now able to interact with another protein, we can get a signal transduction pathway because proteins can now interact with other proteins with which they previously could not.
- Location in which they are found.
49
- Thus, adding the phosphate group to a protein could change its: (3)
- Activity (increase or decrease).
- Interactions with other proteins, because if its shape changes and it is now able to interact with another protein, we can get a signal transduction pathway because proteins can now interact with other proteins with which they previously could not.
- Location in which they are found.
50
- Phosphorylation is very important in signalling pathways, regulating ______ of proteins, and regulation of the cell cycle.
degradation
51
What is Kinase Catalytic Domain? (11)
- Recall that a domain is a portion of a protein that can fold stably into a 3D shape, and it has a particular function.
- The kinase catalytic domain is the domain of an enzyme (which is a protein) that contains the catalytic/active site.
- The catalytic domain folds into a two-lobed structure, like a bean or kidney.
- The upper lobe is the N-terminal lobe (or the amino end), and it is small. Within the N-terminal is an
ATP binding loop that binds ATP.
- The ATP binding loop has a Lysine somewhere in it.
- The lower lobe is the C-terminal lobe (or the carboxyl end), and it is large. The C-terminal has a catalytic loop that binds the target protein.
- The catalytic loop has an Aspartic Acid somewhere in it.
- The cleft between the two lobes is the site of catalysis.
- An activation loop is found in the C-terminal loop, and this blocks the catalytic cleft (where the target protein binds) until the activation loop is phosphorylated.
- The activation loop flips outward when in it is phosphorylated and thus the catalytic loop becomes
available to bind the target protein.
- Once ATP and protein are bound to the kinase catalytic domain, catalysis can occur in the cleft.
52
What is Kinase Catalytic Domain? (11)
- Recall that a domain is a portion of a protein that can fold stably into a 3D shape, and it has a particular function.
- The kinase catalytic domain is the domain of an enzyme (which is a protein) that contains the catalytic/active site.
- The catalytic domain folds into a two-lobed structure, like a bean or kidney.
- The upper lobe is the N-terminal lobe (or the amino end), and it is small. Within the N-terminal is an
ATP binding loop that binds ATP.
- The ATP binding loop has a Lysine somewhere in it.
- The lower lobe is the C-terminal lobe (or the carboxyl end), and it is large. The C-terminal has a catalytic loop that binds the target protein.
- The catalytic loop has an Aspartic Acid somewhere in it.
- The cleft between the two lobes is the site of catalysis.
- An activation loop is found in the C-terminal loop, and this blocks the catalytic cleft (where the target protein binds) until the activation loop is phosphorylated.
- The activation loop flips outward when in it is phosphorylated and thus the catalytic loop becomes
available to bind the target protein.
- Once ATP and protein are bound to the kinase catalytic domain, catalysis can occur in the cleft.
53
- Shown alongside in green is the activation loop before it is phosphorylated. It is blocking the ______ site.
- Shown in ______ is the outward movement of the activation loop after it has been phosphorylated. It exposes the catalytic site, and the kinase is thus active. It can perform its function of phosphorylating a protein.
catalytic
yellow
54
- Shown alongside in green is the activation loop before it is phosphorylated. It is blocking the ______ site.
- Shown in ______ is the outward movement of the activation loop after it has been phosphorylated. It exposes the catalytic site, and the kinase is thus active. It can perform its function of phosphorylating a protein.
catalytic
yellow
55
What are the protein kinases? (3)
- Kinases are usually found either as receptors (they have two jobs, to be a kinase and to be a receptor), or they themselves are not receptors but they are associated with receptors in the membrane, or if they are in the cytoplasm, they phosphorylate receptor proteins or membrane receptors.
- They are found early on in signalling pathways.
- Recall that kinases add a phosphate to Serine, Threonine, or Tyrosine amino acids.
56
- Types of kinases include:
1) Serine/Threonine Protein Kinases
2) Tyrosine Kinases
57
- Types of kinases include:
1) Serine/Threonine Protein Kinases
2) Tyrosine Kinases
58
Types of Protein Kinases:
1) Serine/Threonine Protein Kinases = (3)
- These kinases attach a phosphate to Serine and Threonine.
- Cytoplasmic (found in cytoplasm) Serine/Threonine Kinases include MAPK, AKT, PKA, and PKC.
- Receptor Serine/Threonine Kinases are part of a receptor. The receptor is a protein that also has intrinsic Serine/Threonine Kinase activity. It is both a kinase and a receptor. Examples of Receptor Serine/Threonine Kinases include TGFßRI and TGFßRII (Transforming Growth Factor Beta Receptor 1 and 2). They will phosphorylate their target proteins on Serine and Threonine.
59
Types of Protein Kinases
2) Tyrosine Kinases = (3)
- These kinases attach a phosphate to Tyrosine.
- Tyrosine Kinases can be found inserted in the plasma membrane of the cell and act as receptors. These are called Receptor Tyrosine Kinases, and an example is EGFR (Epidermal Growth Factor Receptor).
- There are also Tyrosine Kinases found in the membrane, but it is a separate protein, and it is associated with another membrane receptor. This means that it itself is not a receptor, but it is associated with another receptor. An important example of this is JAK (Janus Kinase). Cytoplasmic Tyrosine Kinases attach a phosphate to a tyrosine amino acid of a protein found in the cytoplasm. An example of this is SRC.
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Revision:
- Domain
- Subunit
- Domain – A portion of tertiary structure that folds stably into a 3D shape and has its own function. The domain is found within the same polypeptide chain. Alongside is the full tertiary structure of a single protein, and within it are the regions that fold stably. These are the domains, and 3 domains can be seen in the single protein.
- Subunit – A separate polypeptide chain that gives a protein quaternary structure. Alongside is a protein formed by 4 subunits (separate polypeptide chains)
60
Revision:
- Domain
- Subunit
- Domain – A portion of tertiary structure that folds stably into a 3D shape and has its own function. The domain is found within the same polypeptide chain. Alongside is the full tertiary structure of a single protein, and within it are the regions that fold stably. These are the domains, and 3 domains can be seen in the single protein.
- Subunit – A separate polypeptide chain that gives a protein quaternary structure. Alongside is a protein formed by 4 subunits (separate polypeptide chains)
61
How are kinases regulated? (3)
- Kinases may have a regulatory (inhibitory) domain. This is part of the same polypeptide chain that contains the catalytic domain. Thus, this kinase would have at least 2 domains, a bean-shaped catalytic domain as explained previously, and a regulatory (inhibitory) domain. Examples of kinases that have a regulatory domain include Calcium Calmodulin Protein Kinase and SRC Tyrosine Kinase.
- Kinases may have a regulatory subunit. This is another polypeptide that binds to the kinase polypeptide/protein and regulates it. It masks the active site when bound. An example of this is PKA (Protein Kinase A).
- Kinases may need another protein to bind to the kinase. This activates the kinase. An example of a kinase that is activated by the binding of another protein is CDK (Cyclin Dependent Kinase), which requires the binding of Cyclin.
62
Examples of Kinases: (2)
1) Serine/Threonine Kinases
2) Tyrosine Kinases
63
- Calcium Calmodulin Dependent Kinase
- As shown above, the regulatory domain is interacting with the catalytic domain, and is inhibiting it. This kinase is thus _______.
- When Calcium binds to Calmodulin, Calcium Calmodulin is ______.
- When Calcium Calmodulin binds to the regulatory domain, it will no longer interact with the _____ ______, and thus the Calcium Calmodulin Dependent kinase is active, as shown below.
inactive
formed
catalytic domain
64
- Protein Kinase A (PKA)
- PKA is a _______ (has 4 subunits) kinase.
- Two subunits are regulatory subunits, and two are catalytic subunits.
- _____ (cyclic Adenosine Monophosphate) is produced in a _________ pathway, and it binds to the regulatory subunits and changes their shape, causing them to dissociate and move away from the catalytic subunits.
- The catalytic subunits would now be active, and the kinase can _______ its target protein.
tetrameric
cAMP
signalling
phosphorylate
65
What is Cyclin Dependent Kinase (CDK)? (3)
- This kinase plays an important role in regulation of the cell cycle.
- CDK is a small protein composed of little more than a catalytic domain.
- Cyclin Dependent Kinase (CDK) is activated by binding of a cyclin.
66
What are Receptor Tyrosine Kinases? (2)
- Receptor Tyrosine Kinases are found in the membrane and the protein is both a receptor, and a
Tyrosine Kinase.
- Inactive Receptor Tyrosine Kinases are found as monomers in the cell membrane.
67
- Receptor Tyrosine Kinases
- Each monomer has three domains: (3)
1) An extracellular domain, to which a ligand (small molecule with transmits signals between cells) will bind.
2) A transmembrane domain that passes through the membrane.
3) An intracellular domain that has a catalytic domain (shown in red).
68
- Receptor Tyrosine Kinases
- Each monomer has three domains: (3)
1) An extracellular domain, to which a ligand (small molecule with transmits signals between cells) will bind.
2) A transmembrane domain that passes through the membrane.
3) An intracellular domain that has a catalytic domain (shown in red).
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What happens in the process of Receptor Tyrosine Kinase? (5)
- Below the catalytic domain is a tail of tyrosine amino acids, which are in a sequence motif (a sequence motif is the tyrosine amino acid and the other amino acids around it, and this sequence is recognised by the kinase).
- When the ligand binds, dimerization occurs, which is the combining of two monomers to form a dimer.
- Due to interaction between the monomers and the ligand, we get a change in shape (conformational shape), which as you should recall, always means a change in function.
- ATP can now access the active site in the catalytic domain and the Tyrosine kinase becomes active.
- The Tyrosine Kinase now phosphorylates Tyrosine (in the tail below the catalytic site). The left catalytic site (shown in red) phosphorylates a Tyrosine on the left tail, and the right catalytic site phosphorylates a Tyrosine on the right tail. We say the Tyrosine Kinase autophosphorylates itself on the cytoplasmic tail Tyrosine amino acids.
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- SRC Tyrosine Kinase:
- SRC Tyrosine Kinase is found in the cytoplasm.
- It has 3 domains, 1 ______ domain, and 2 ______
domains
– SH2 and SH3 (shown in yellow and green in
the image).
- The SH2 domain can be seen interacting with the ____ of the protein.
- The SH3 domain can be seen interacting with and
_______ the catalytic domain.
- The two inhibitory/regulatory domains are interacting with and inhibiting the catalytic domain, and thus the SRC Tyrosine Kinase is _______.
catalytic
regulatory
tail
inhibiting
inactive
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How is SRC Tyrosine Kinase activated?
The phosphate on the tail which was interacting with the SH2 domain must be removed.
A protein (shown in orange) comes and binds to the SH2 domain and moves it away from the catalytic domain. Phosphorylation of the activation loop must occur, and the activation loop will flip outward from the catalytic cleft.
- The SRC Tyrosine Kinase is now active and can Phosphorylate its target Tyrosine.
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Structure:
- You do not need to know their full structure – simply see the side chains (attached to central C).
- Note that three amino acids that can be phosphorylated by kinases all contain OH.
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What is Phosphoprotein Phosphatases? (2)
- These are enzymes that dephosphorylate (remove phosphate) proteins that have been phosphorylated.
- They catalyse dephosphorylation.
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- Unlike kinases that developed from a common ancestor, phosphatases have evolved from separate families. Phosphatases are completely separate enzymes.
- Similarly to how we have 2 types of kinases (Serine/Threonine Kinases and Tyrosine Kinases), we have 2 phosphatases:
1) Phosphoserine/Phosphothreonine Phosphatases
2) Phosphotyrosine Phosphatases
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1) Phosphoserine/Phosphothreonine Phosphatases
- These are ________ (subunits are different to each other) proteins consisting of a catalytic subunit associated with regulatory subunits (they consist of more than one subunit).
- An example is _____ which regulates cell proliferation by removing the phosphate from proteins in signalling pathways that have been activated by growth factors.
- Another example is _____ (Calcineurin) which has a regulatory subunit that binds ______ (Ca2+)
heteromeric
PP2A
PP2B
Calcium
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2) Phosphotyrosine Phosphatases:
- There are about ______ protein Tyrosine Phosphatases.
- They have very specific substrates.
- There are two main classes of Phosphotyrosine Phosphatases:
1) ______ (such as PTP1B – Phosphotyrosine Phosphatase 1 B).
2) _____-______ which are proteins within the membrane (such as CD45 Leucocyte Common Antigen).
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Intracellular
Receptor-linked
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What is Intercellular Communication? (3)
- Intercellular communication is communication between cells.
- The billions of cells in our body requires communication so that they receive the right information at the right time so that they can work together.
- Without proper communication, cells will not function correctly.
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What is 1) Autocrine Signalling – Same cell?
- A cell secretes signalling molecules, and these signalling molecules bind to cell surface receptors on the same cell that produced them.
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What is 2) Paracrine signalling – Local cells? (2)
- A cell releases signalling molecules into the extracellular space, and these signalling molecules act locally on neighbouring cells.
- These signalling molecules are rapidly degraded so that the signal does not travel further away.
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What is 2) Paracrine signalling – Local cells? (2)
- A cell releases signalling molecules into the extracellular space, and these signalling molecules act locally on neighbouring cells.
- These signalling molecules are rapidly degraded so that the signal does not travel further away.
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What is 3) Endocrine signalling – Far away cells? (2)
- Endocrine cells secrete hormones into the bloodstream that are then distributed widely throughout the body.
- The target cell is usually far from the endocrine cell.
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What is 4) Contact-dependent signalling/ Cell to Cell signalling? (2)
- This is signalling between 2 (usually epithelial) cells.
- There is cell membrane to cell membrane contact, as cells need to be in direct contact with each other.
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What is 5) Synaptic signalling?
- This is signalling performed by neurons that transmit signals electrically along their axons and release neurotransmitters at synapses, which are often located far away from the cell body.
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Why do cells need to communicate? (4)
- Cells need signalling for normal function. An example is for movement – your muscles have to co- ordinate their contractions.
- Cells need signalling to tell them when to grow and divide (proliferation). This is required in the developing foetus as well as in wound healing.
- Cells need signalling for differentiation (to become specialised). An example is stem cells in the bone marrow that differentiate into blood cells.
- Cells need to be signalled when to die – to commit suicide. If a cell is a threat to the organism such as cancerous cells or cells with damaged DNA, it must die so that it cannot harm the cell. Programmed cell death is called apoptosis.
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Why do cells need to communicate? (4)
- Cells need signalling for normal function. An example is for movement – your muscles have to co- ordinate their contractions.
- Cells need signalling to tell them when to grow and divide (proliferation). This is required in the developing foetus as well as in wound healing.
- Cells need signalling for differentiation (to become specialised). An example is stem cells in the bone marrow that differentiate into blood cells.
- Cells need to be signalled when to die – to commit suicide. If a cell is a threat to the organism such as cancerous cells or cells with damaged DNA, it must die so that it cannot harm the cell. Programmed cell death is called apoptosis.
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What is Intracellular Receptor (Hydrophobic Signalling)? (2)
- This is signalling where the signalling molecule is hydrophobic (water-hating).
- If it is hydrophobic, it must mean it is a lipid, and these are usually steroid hormones. They can also
be thyroid hormones (contain tyrosine) and some vitamins.
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- Hydrophobic signalling consists of 3 components:
- Ligands (the extracellular signalling molecule).
- Receptors.
- Transcription factors.
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- Recall that the membrane of a cell is made of lipids, and so too is the steroid ligand. The steroid ligand can thus diffuse through the membrane. The steroid ligand enters the cell and binds to a specific intracellular receptor inside the cell.
- The ligand and receptor together form a transcription factor-like protein called the receptor- hormone complex, which moves into the nucleus, binds to ta hormone-response element (resembles enhancer elements), and results in the production of specific proteins.
- Hydrophobic signalling results in the production of new signalling because we have initiated transcription and translation.
- Learn the following: (5)
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What is Membrane Receptor (Hydrophilic signalling)? (5)
- This is signalling where the signalling molecules are hydrophilic (water-loving).
- These molecules are soluble in fluid, and thus cannot pass through our lipid membrane.
- Hydrophilic signalling molecules are usually protein or polypeptide based.
- Thus, the protein/polypeptide ligands bind to specific receptors present on the cell membrane.
- That transmembrane receptor converts the extracellular signal into an intracellular chain of
biochemical events which amplifies the signal.
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Membrane Receptor (Hydrophilic signalling)
- For this signalling pathway, information is conveyed through the cytoplasm through one of two
ways:
- Through diffusible elements (such as Calcium of cAMP) called second messengers.
- Through protein-protein interactions via their domains resulting in signal transduction.
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- Hydrophilic signalling pathways result in the activation of a _____ _______.
- The final resulting outcome of signalling through a hydrophilic signalling pathway is proteins with
altered function, or transcription factors are activated, resulting in the production of new proteins.
- In hydrophobic signalling, new proteins are produced, not proteins with ______ function.
tyrosine kinase
altered
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What is the Interaction Domains in Signal Transduction?
- Different proteins interact through their domains. The domains of one protein interact with the domains of another.
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Interaction Domains in Signal Transduction
- Different proteins interact through their domains. The domains of one protein interact with the domains of another.
- There are approximately 40 to 50 different domains. We will focus on a few: (4)
- DNAB – DNA Binding domain. These Domains bind to DNA and an example of where a DNA Binding domain can be found is in transcription factors.
- Catalytic Domain – We have covered this already.
- PH – Plecktrin homology domain – This domain binds to PI-3,4,5-trisP (Phosphoinositol-3,4,5- trisPhosphate), which is a phospholipid present in the cell membrane.
- SH2 – SRC homology 2 domain – SH2 binds to a phosphorylated Tyrosine. Note that it is not just the amino acid Tyrosine, it is the amino acids around the Tyrosine called the peptide motif that it binds to. The SH2 binds to a peptide motif containing a phosphorylated Tyrosine.
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Hydrophilic signalling process: (8)
- An extracellular signalling molecule called a ligand (usually a protein or polypeptide) binds to the membrane receptor.
- The receptor transduces the signal into the cell where further signalling will occur.
- An adaptor protein links the membrane receptor to other proteins in the cell cytoplasm, such as a
monomeric G protein.
- Heterotrimeric G proteins link directly to the membrane receptor.
- The Heterotrimeric G protein results in the production of a second messenger.
- The second messenger or the monomeric G protein can activate a kinase.
- The kinase can then phosphorylate another protein and alter its function, or it can phosphorylate a
transcription factor to activate it that activated transcription factor will move into the nucleus and bind to the promoter of genes. We will then get transcription of those genes and a new protein will be made.
- Scaffold proteins can also be found in signalling pathways, and they bind numerous proteins together and increase the efficiency of signalling in that signalling pathway.
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Components of Hydrophilic Signalling: (8)
1) Ligands
2) Membrane Receptors
3) Adaptors
4) G Protein
5) Second Messengers
6) Protein Kinases
7) Scaffold Proteins
8) Transcription Factors
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Components of Hydrophilic Signalling:
1) Ligands: (3)
- Ligands are extracellular signalling molecules that bind to specific receptors.
- They are known as 1st messengers or hormones.
- Examples include chemokines (signalling molecules that cause movement of cells), cytokines
(signalling molecules used between cells of the immune system), growth factors (signalling molecules that cause proliferation of cells), and hormones (signalling molecules that results different physiological function effects related to the specific hormone).
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Components of Hydrophilic Signalling
2) Membrane Receptors: (4)
- If a receptor is not present, the ligand will not bind to it, and we will not get a signal.
- The response depends on the relative numbers of receptors for that hormone or in the target cells,
and the affinity (strength) of the bond between the hormone and the receptor. A greater afiinity
will result in a greater signal.
- Hydrophilic ligand receptors have extracellular domains that interact with the specific ligand (recall
that this is because the ligand is hydrophilic and cannot pass through the membrane, and so the
receptor must have a piece of itself outside of the cell).
- Hydrophilic ligand receptors have domains/motifs (a sequence of amino acids) in the cytoplasm
that can transduce the signal from outside to inside because the domain/motif interacts with
intracellular signalling molecules.
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- There are three main types of membrane receptors: (3)
1) Those without kinase activity, such as GPCR (G Protein Coupled Receptors).
2) Those with intrinsic kinase activity (the receptor protein itself is a kinase), such as Receptor Tyrosine Kinase.
3) Those associated with a kinase (this receptor is bound to a separate protein that is a kinase), such as Cytokine receptors, T Cell Receptors, and B Cell Receptors.
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Components of Hydrophilic Signalling
3) Adaptors: (3)
- Adaptors are relatively small proteins with usually no more than two or three domains whose function is to link two proteins together.
- Quite often, adaptor proteins have an SH2 domain (recall that SH2 domains bind to phosphorylated Tyrosine amino acids). Different SH2 domains recognise the P-Tyrosine (phosphorylated Tyrosine) associated with different amino acids forming the sequence motifs.
- Shown alongside is an adaptor protein with 3 domains. The SH2 domain will bind to a P-Tyrosine and the amino acids around it, and one of the other domains will bind to the next protein in that signalling pathway.
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What is Adaptor functioning? (3)
- A receptor is phosphorylated on a Tyrosine amino acid.
- An adaptor protein with an SH2 domain comes along and binds to the P-Tyrosine and the amino
acids surrounding it.
- Other domains of the adaptor protein interact with domains of the next protein in that signalling
pathway.
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What are G proteins? (5)
- G proteins get their name from the fact that they bind GTP (Guanosine Trisphosphate) or GDP (Guanosine Diphosphate). If it bound to ATP, it would be called an A protein.
- When proteins are bound to GDP, they are inactive.
- G proteins are activated by the exchange of GDP for GTP (G proteins are active when GTP is bound).
Note that GDP does not get changed into a GTP. The entire GDP is replaced by a GTP.
- If G proteins are active, they can pass the signal on.
- G proteins have intrinsic GTPase activity. This means that they can convert GTP back to GDP by
removing the terminal phosphate. In doing so, they switch themselves off. They are no longer
active, and the signalling pathway is stopped.
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- There are two types of G proteins:
1) Small Monomeric (one subunit) such as
RAS.
2) Heterotrimeric (three different subunits) G Protein
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G protein:
1) Small Monomeric (one subunit) such as
RAS = (2)
- Signalling through hydrophilic pathways recruits a GEF (Guanine Exchange Factor) which exchanges GDP for GTP on the monomeric G protein RAS. GDP is released. This activates RAS.
- RAS’s intrinsic GTPase activity converts GTP back to GDP by removing the terminal phosphate of GTP. RAS is now bound to GDP and no longer active.
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G protein:
2) Heterotrimeric (three different subunits) G Protein = (8)
- Consists of an α subunit, a ß subunit, and a γ subunit.
- The Alpha (α) subunit binds GTP/GDP.
- If GDP is bound, the heterotrimeric G protein is inactive.
- When a ligand (such as a hormone) binds to the receptor (called a G Protein coupled receptor,
because heterotrimeric G proteins link directly to the receptor) changes shape and the GDP is released and a GTP is attached to the α subunit. The G protein is now
active.
- The trimer dissociates into an α subunit with the GTP attached, and the ß and γ subunit.
- Recall that G proteins have intrinsic GTPase activity. The α subunit can convert the GTP back to GDP by removing the terminal/gamma phosphate from the GTP.
- This produces an α subunit with GDP that is inactive.
- The ß and γ reassociate with the α subunit, and we are back to having our inactive heterotrimeric G protein.
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G protein:
2) Heterotrimeric (three different subunits) G Protein = (8)
- Consists of an α subunit, a ß subunit, and a γ subunit.
- The Alpha (α) subunit binds GTP/GDP.
- If GDP is bound, the heterotrimeric G protein is inactive.
- When a ligand (such as a hormone) binds to the receptor (called a G Protein coupled receptor,
because heterotrimeric G proteins link directly to the receptor) changes shape and the GDP is released and a GTP is attached to the α subunit. The G protein is now
active.
- The trimer dissociates into an α subunit with the GTP attached, and the ß and γ subunit.
- Recall that G proteins have intrinsic GTPase activity. The α subunit can convert the GTP back to GDP by removing the terminal/gamma phosphate from the GTP.
- This produces an α subunit with GDP that is inactive.
- The ß and γ reassociate with the α subunit, and we are back to having our inactive heterotrimeric G protein.
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Components of Hydrophilic Signalling
5) Second Messengers: (3)
- Activation of heterotrimeric G proteins (with GTP attached) results in activation of effector enzymes.
- Effector enzymes (proteins that catalyse reactions and produce products) produce second messengers, which are diffusible chemicals such as cAMP and IP3 (Inositol Trisphosphate).
- These second messengers then activate kinases.
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Components of Hydrophilic Signalling
6) Protein Kinases: (4)
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Components of Hydrophilic Signalling
7) Scaffold Proteins: (5)
- Scaffold proteins are large proteins that can bind various other proteins to bring them together in a complex.
- Scaffold proteins have numerous domains that can bond many other proteins to them.
- Scaffold proteins have no enzymatic activity.
- They can be phosphorylated at various sites and thus recruit many different proteins with SH2
domains (recall that SH2 domains only bind to Tyrosine amino acids that have been phosphorylated).
- Because we have several proteins now bound together, we can increase the efficiency of signalling
through signalling pathways.
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Components of Hydrophilic Signalling
8) Transcription Factors: (5)
- Recall that transcription factors are proteins that bind to promoters of genes and either increase or decrease transcription of a gene.
- The promoter contains several conserved consensus sequences (such as TATA and CAAT) which bind general transcription factors, as well as other specific transcription factors to specific sequences within those promoters.
- Specific transcription factors give specificity to the signal. They determine whether those genes are expressed or not.
- Genes with promoters with the same sequences will bind the same transcription factors, giving a combined response.
- We need specific transcription factors as well as general transcription factors bound to initiate transcription of a gene.
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Speed of Signalling Pathways
- Hydrophobic signalling has far less components that Hydrophilic signalling.
- Which is faster depends on what is happening.
- Recall that hydrophobic signalling only produces new proteins, whereas hydrophilic signalling can
______ proteins, or produce new proteins.
- If a protein is being altered, it will be a very quick process, as the protein already exists.
- If a new protein is being produced, it will be a lengthy process as transcription and _________ take time.
alter
translation
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Speed of Signalling Pathways
- Hydrophobic signalling has far less components that Hydrophilic signalling.
- Which is faster depends on what is happening.
- Recall that hydrophobic signalling only produces new proteins, whereas hydrophilic signalling can
______ proteins, or produce new proteins.
- If a protein is being altered, it will be a very quick process, as the protein already exists.
- If a new protein is being produced, it will be a lengthy process as transcription and _________ take time.
alter
translation
