C. Protein Analysis Tools Flashcards
- Protein ______ tools and techniques enable one to separate proteins and subsequently identify them.
analysis
- Separation and identification methods include: (4)
- SDS-PAGE
- 2D-Gel Electrophoresis
- Isoelectric focusing (IEF)
- Western blot
- Separation and identification methods include: (4)
- SDS-PAGE
- 2D-Gel Electrophoresis
- Isoelectric focusing (IEF)
- Western blot
- 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.
chromatography
insulin
What does SDS-PAGE stand for?
- SDS-PAGE stands for Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis.
How does SDS-PAGE work? (5)
- 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.
How does SDS-PAGE work? (5)
- 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.
What is SDS? (3)
- 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.
What happens during PAGE? (2)
- 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.
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.
Negatively Proportional
Rf
What is 2D-Gel Electrophoresis? (3)
- 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.
2D-Gel Electrophoresis
What are the steps involved? (2)
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.
2D-Gel Electrophoresis
What are the steps involved? (2)
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.
What is Isoelectric Focusing (IEF)? (8)
- 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.
What is Protein Purification: Chromatography?
- 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.
What is Protein Purification: Chromatography?
- 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.
There are 3 main types of chromatography, based on the protein that is the basis of the purification: (3)
1) Affinity-Based Chromatography
2) Size-Exclusion Chromatography
3) Ion-Exchange Chromatography
What is 1) Affinity-Based Chromatography?
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.
What is 1) Affinity-Based Chromatography?
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.
What is 2) Size-Exclusion Chromatography?
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.
What is 3) Ion-Exchange Chromatography?
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.
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.
affinity-purified
What is the Recombinant DNA (rDNA)?
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).
What is the Recombinant DNA (rDNA)?
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).
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
Cloning Expression Cassettes
- Cloning expression cassettes enables protein expression and is illustrated as follows: (5)
- The gene responsible for the protein is isolated from the DNA of a human cell using restriction enzymes.
- 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.
- 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.
- 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.
- Note that in this example plasmid was used as a vector. There are other vectors that can be used.
Cloning Expression Cassettes
- Cloning expression cassettes enables protein expression and is illustrated as follows: (5)
- The gene responsible for the protein is isolated from the DNA of a human cell using restriction enzymes.
- 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.
- 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.
- 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.
- Note that in this example plasmid was used as a vector. There are other vectors that can be used.
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
What are the advantages or challenges of each?
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.
What are Cloning Vectors?
- Cloning vectors have important features that enable them to play their role as carries of DNA fragments/genes of interest.
- 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.
- 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.
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.
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.
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.
- 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.
- 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.
The Development of Recombinant Insulin: (5)
*cDNA is complementary DNA.
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.
Area of treatment:
Example of protein:
- 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
- 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
- For kinases:
Substrates Products
Target Protein + ____ ➔ Target Protein-P + ____
ATP
ADP
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
What happens when kinases are added to this reaction?
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.
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.
- 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
- 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
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)
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)
- 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
- 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
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.
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.
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.
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.
What is Phosphoprotein Phosphatases? (2)
- These are enzymes that dephosphorylate (remove phosphate) proteins that have been phosphorylated.
- They catalyse dephosphorylation.
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.
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.
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.
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.
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.
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.
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.
- 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)
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.
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.
- 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.
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.
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.
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.
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.
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.
Components of Hydrophilic Signalling
6) Protein Kinases: (4)
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
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
