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(c) Protein structure, ligand binding and conformational change
(c) Protein structure, ligand binding and conformational change
Let's begin our journey into protein structure by watching this fantastic introductory video. Just click on the pink button "What is a protein?" below.
Let's begin our journey into protein structure by watching this fantastic introductory video. Just click on the pink button "What is a protein?" below.
There are 4 levels of protein structure
There are 4 levels of protein structure
Let's explore PRIMARY structure
Let's explore PRIMARY structure
Amino acid sequence determines protein structure
Amino acid sequence determines protein structure
Proteins are polymers of amino acid monomers.
Proteins are polymers of amino acid monomers.
In Eukaryotes, the DNA sequence is transcribed into RNA within the nucleus of a cell. After RNA splicing, the mature RNA transcript moves to ribosomes, where it undergo translation. This happens when tRNA molecules bring individual amino acid monomers based on complementary codon-anticodon pairing.
As individual amino acid monomers are produced at the ribosome, peptide bonds form between them, creating a polypeptide chain. This linear sequence of amino acids forms the primary structure of a protein. This is depicted in the diagram (right).
Amino acids have the same basic structure, differing only in the R group present.
Amino acids have the same basic structure, differing only in the R group present.
An amino acid has both amino (-NH2) and carboxyl (-COOH) groups at either end. These are known as the N-terminus (NT) and C-terminus (CT), respectively. The R-group (side chain) is the part of the amino acid that is variable and determines how the protein will fold and interact.
Task 24
Task 24
The theory was really cranking up a notch now and wee Jonny stopped swinging on his seat. He craned his neck to see what his special buddy Elin was writing. She was sketch the basic structure of an amino acid in her notes. Jonny did the same. You should too.
Jonny's sketch is available here.
Amino acids are linked by peptide bonds to form a polypeptide.
Amino acids are linked by peptide bonds to form a polypeptide.
A condensation reaction (see below) joins 2 amino acids together to form a peptide bond, with the release of a water molecule.
A condensation reaction (see below) joins 2 amino acids together to form a peptide bond, with the release of a water molecule.
This short video provides a lovely summary of protein structure, perfectly highlighting some of the key points for your course.
Task 25
Task 25
Big Davie was loving this bit of the course - biochemistry was his thing and he liked how his learning in Chemistry was helpful in Biology too. Knowing that he needed to recognise the chemical structure of a peptide bond, he drew a diagram of 3 amino acids joined via peptide bonds. Time for you to follow Big Davie's footsteps.
Big Davie's sketch is available here.
R groups of amino acids vary in size, shape, charge, hydrogen bonding capacity and chemical reactivity. The wide range of functions carried out by proteins results from the diversity of R groups. Amino acids are classified according to their R groups:
Basic (positively charged)
Acidic (negatively charged)
Polar
Hydrophobic
Task 26
Task 26
Hannah felt a bead of sweat on the back of her neck: "Miss, are you tellin' us we need to, like, memorise all they mad wee amino acid structures?!"
"Nope, I definitely didn't just say that Hannah. You definitely do NOT need to know the names or the structures of individual amino acids. However, it is useful for you to highlight key elements of R groups that allow classification into the 4 main types of amino acids".
To help with this, Dr McRobbie asked the class to name and draw an example of:
A hydrophobic/non-polar amino acid
A polar amino acid
A basic/positively-charged amino acid
An acidic/negatively-charged amino acid
You should now do the same.
Suggested answers are available here.
Task 27
Task 27
To really push the class further, Dr McRobbie asked them all to draw a short polypeptide consisting of a hydrophobic, polar, basic and acidic amino acid. She highlighted that this polypeptide would contain 3 peptide bonds. "Och, Miss, you're pushing yer luck now aren't ye?" joked Big Davie. Secretly, he was loving it.
You should attempt this task. Suggested answer is included here.
Task 28
Task 28
As a quick refresher on translation, the class picked up an old task they had first encountered in S3. The task involved using a basic Codon Wheel to work out the chain of amino acids from a DNA sequence. The translated sequence could then be matched up to a protein on the "Function Finder" cards to hammer home how the order of bases affects the order of amino acids and therefore the structure and function of a protein.
As a quick recap, complete this task. You will need to use the Codon Wheel (right) to work out the chain of amino acids and the "Function Finder" cards to match your translated sequence to a particular protein - there are 12 proteins so remember to scan through until you find your one!
Answers are available here.
To decode a codon, find the first letter of your sequence in the inner circle and work outwards to see the corresponding amino acid. For example: CAT codes for H (histidine).
protein_profiles.pdfLet's explore SECONDARY structure
Let's explore SECONDARY structure
Secondary structure includes the hydrogen bonding along the backbone of the protein strand results in regions of secondary structure.
Secondary structure includes the hydrogen bonding along the backbone of the protein strand results in regions of secondary structure.
This includes:
This includes:
> alpha helices
> alpha helices
> parallel or anti-parallel beta-pleated sheets
> parallel or anti-parallel beta-pleated sheets
> turns.
> turns.
The link above discusses secondary structure extremely well from 2min 10s until 6min 50s.
An anti-parallel beta pleated sheet
An anti-parallel beta pleated sheet
A section of a protein showing an alpha helix (blue) and an anti-parallel beta sheet (yellow/red). Loop regions are shown in grey connecting these areas.
A section of a protein showing an alpha helix (blue) and an anti-parallel beta sheet (yellow/red). Loop regions are shown in grey connecting these areas.
Task 29
Task 29
"Well Miss, you shouldv'e told us that we were really studying Art when we picked this subject", mocked Big Davie from the back of the class.
"Well, Biology is a wonderfully varied subject that allows you to express yourself in all manner of ways in here, doesn't it Big Davie? Now, get on with the next piece of Artwork and express yourself by drawing 2 polypeptide chains of 3 amino acids long in a manner that would allow a beta sheet to form".
Big Davie got on with it. He did a superb job. Predict what was on his page by attempting the task yourself. His work is available here.
Let's explore TERTIARY structure
Let's explore TERTIARY structure
The polypeptide folds to give rise to a tertiary structure. This conformation is stabilised by interactions between R groups, including:
The polypeptide folds to give rise to a tertiary structure. This conformation is stabilised by interactions between R groups, including:
- Hydrophobic interactions
- Ionic bonds
- London dispersion forces
- Hydrogen bonds
- Disulfide bonds - these are strong covalent bonds between R groups that contain sulfur.
The link above is an excellent demonstration of tertiary structure - start at 1min 10s.
Task 30
Task 30
"So, Hydrogen bonds are the only type of interaction stabilising Secondary structure but there are many more stabilising a protein in its tertiary form?" considered Olivia.
"Yes, you are absolutely correct". Dr McRobbie replies. "Take a look at this diagram below showing the tertiary structure of a protein. Can you identify what type of interactions are stabilising the structure at each point"?
What do you think Olivia wrote down as her answers for interactions A to E?
Suggested answers are here.
Let's explore QUATERNARY structure
Let's explore QUATERNARY structure
Quaternary structure exists in proteins with two or more connected polypeptide subunits.
Quaternary structure exists in proteins with two or more connected polypeptide subunits.
Quaternary structure describes the spatial arrangement of the subunits.
Quaternary structure describes the spatial arrangement of the subunits.
This diagram shows the tertiary structure of a protein on the left. The tertiary structure shows one subunit. However, in its final form, this protein has quaternary structure, consisting of two subunits (shown in different shades of green, right).
Task 31
Task 31
"Oh yaaaas, Miss, we going on the Chromebooks for this period, aye?" Big Davie had entered the room. David, Olivia and Kasia followed closely behind and were clearly looking forward to not having to think too hard this period.
"Nope, not the full period Big Davie...but, yes, for a part of it. I would like you to carry out a quick piece of research to find a protein (other than haemoglobin) that has quaternary structure. I would like you to either sketch the protein into your notes or find a suitable electronic image of the protein (must use a good source, e.g. Protein Data Bank - not wikipedia) and then find out how many subunits are in the quaternary structure of the protein".
"Right, so definitely not this new game I just heard about Miss?"
Big Davie was chancing his luck this period. Could you carry out the research too in case he doesn't stump up the good! Some useful links have been included below.
Suggested answers are shown here.
Haemoglobin (shown above) is an example of a protein with quaternary structure.
Prosthetic groups
Prosthetic groups
A prosthetic group is a non-protein unit tightly bound to a protein and is necessary for its function.
Haemoglobin has a prosthetic group (haem), which is responsibility for the protein's ability to bind oxygen.
RasMol Activity
RasMol Activity
I've written a short activity using RasMol. This is a protein visualisation programme that you can use with any protein structure. Follow the instructions in the tutorial to play around with some of the details you have been learning so far in Key Area 2c.
Before looking at some past paper questions, watch this summary video from Mrs Kennedy.
Effect of temperature and pH on protein structure
Effect of temperature and pH on protein structure
We are know from early secondary school that temperature and pH affect enzyme activity. We have learned that extremes of temperature and pH values distant from the enzyme's optimum range can result in denaturation. But why?
Interactions of the R groups can be influenced by temperature and pH
Increasing temperature disrupts the interactions that hold the protein in shape; the protein begins to unfold, eventually becoming denatured.
The charges on acidic and basic R groups are affected by pH. As pH increases or decreases from the optimum, the normal ionic interactions between charged groups are lost, which gradually changes the conformation of the protein until it is denatured.
Task 32
Task 32
Denaturation is something the class had been familiar with since early Secondary school. But now it was slightly different; now they have to understand it in terms of bonding and disruption of interactions. Can you delve deep and explain why changes in temperature or pH can disrupt a protein structure. Be specific to the type of bonds influenced in each case.
Suggested answers are here.
Ligand binding changes the conformation of a protein
Ligand binding changes the conformation of a protein
A ligand is a substance that can bind to a protein. This might be used to:
activate receptors
activate/inhibit enzymes
substrate binding to enzymes
open and close protein channels
organise other molecules like DNA
to control transcription.
R groups not involved in protein folding can allow binding to ligands.
Binding sites will have a complementary shape and chemistry to the ligand.
As the ligand binds to a protein-binding site, the conformation of the protein changes. This change in conformation causes a functional change in the protein.
Task 33
Task 33
"Time to sharpen those pencils again troops", said Dr McRobbie. "Can you please draw a fairly simple diagram of a ligand bound to a protein and, if you can, indicate possible bonding between R groups on the protein and molecules on the ligand".
Dr McRobbie explained that understanding more about the interactions between ligands and proteins is really important in the context of drug design against diseases. If we know how a protein binds to a ligand, we can potentially disrupt this interaction.
Suggested answers are here.
Allosteric proteins
Allosteric proteins
We have met allosteric sites before during Higher studies of Biology. In general, you have probably heard about this with respect to enzymes, e.g. during your prior studies in regulation of Respiration.
Crystal structural of Phosphofructokinase, a key regulatory allosteric enzyme during glycolysis, is shown to the right.
Task 34
Task 34
Dr McRobbie was loading a video. Jonny was using all his energy to will it to be a 50 min video. Dr McRobbie never showed 50 minute movies. In all likelihood, it would be less than 5 minutes with a pretty annoying voice-over. Still, he stopped swinging in his chair just to focus all his hopeful energy on a feature-length. His hopes were dashed: "Right guys, let's watch this carefully for a couple of minutes. I will then ask you to write down what you think is meant by an "allosteric enzyme".
Based on your learning from Higher and the information in this video, can you complete the task too?
Answers are here.
Allosteric Enzymes
Allosteric Enzymes
Allosteric enzymes contain a second type of site, called an allosteric site. Modulators regulate the activity of the enzyme when they bind to the allosteric site. Following the binding of a modulator, the conformation of the enzyme changes and this alters the affinity of the active site for the substrate.
The binding of a substrate molecule to one active site of an allosteric enzyme increases the affinity of the other active site for binding of subsequent substrate molecules. This is of biological importance because the activity of allosteric enzymes can vary greatly with small changes in substrate concentration.
Positive modulators
Positive modulators
A ligand (modulator) might bind to the allosteric site of an enzyme, causing a conformational change that increases the affinity of the enzyme for the substrate. The rate of the reaction increases and more product is formed.
Negative modulators
Negative modulators
A ligand might bind to the allosteric site of an enzyme, causing a conformational change that decreases the affinity of the enzyme for the substrate. No product is formed.
Task 35
Task 35
The Chromebooks were out again and the room was filled with happy faces. Dr McRobbie loved a bit of bioinformatics and today was another chance to explore these online database.
"Right team, Aspartate transcarbamoylase is an allosteric enzyme of biological importance. Can you please use bioinformatic tools and other online sources to find out about this enzyme. You should access "Expasy" (click the pink button below to access) and, in "Query all databases", type: "aspartate transcarbamoylase". You should then click on the UniProt hits and then select the top Human hit (as shown below: P27708 CAD protein)." Dr McRobbie began.
"Give a general overview of the protein but also consider the key questions below"
Questions to consider:
With respect to this protein, what does it mean to be allosteric?
How is this protein allosterically regulated?
What did this form of regulation mean in the wider context of the cell?
Suggested answers are here.
Allosteric proteins
Allosteric proteins
Many different types of protein molecules exhibit allosterism, not just enzyme molecules. Many allosteric proteins consist of multiple subunits (i.e. they have quaternary structure). Allosteric interactions occur between spatially distinct sites. Allosteric proteins with multiple subunits show cooperativity in binding, in which changes in binding at one subunit alter the affinity of the remaining subunits.
Haemoglobin: an allosteric protein
Haemoglobin: an allosteric protein
Haemoglobin is an example of an allosteric protein. It has quaternary structure and the binding and release of oxygen in each subunit shows cooperativity.
Task 36
Task 36
Watch the following clip from the Wellcome Trust to answer the following questions:
1. Explain what is meant by "cooperativity".
2. Describe and sketch the "Oxygen Dissociation Curve".
3. State one condition that affects haemoglobin's affinity for oxygen.
The affinity of a protein for a ligand is strongly influenced by temperature and pH.
In the case of haemoglobin, small changes in temperature and pH affect the oxygen saturation of the protein (see graph opposite):
Temperature - as temperature increases, oxygen affinity decreases. Temperatures usually increase as respiratory demand increases in the demand; as a consequence, cells and tissues require higher concentrations of oxygen. Haemoglobin must readily dissociate from oxygen under these conditions.
pH - as pH decreases (i.e. as carbon dioxide concentration in the blood increases), the affinity of haemoglobin for oxygen decreases.
As the affinity of haemoglobin for oxygen decreases in actively respiring tissues, oxygen molecules are released from the protein. The promotes increased oxygen delivery to tissues.
Task 37
Task 37
Elin skipped into class, cheerful and happy as always. She was in a chatty mood, Dr McRobbie could tell. She needed to do something quick. "Before you all get too comfy, I'd like you to reflect back on the work we have done on the haemoglobin dissociation curve. We chatted a bit about how this would shift under changing temperature and pH levels. In 3 minutes, I am going to ask someone to come up and sketch the haemoglobin dissociation curve when temperature and pH increase or decrease. I'd also like you to explain the physiological importance of temperature and pH on the binding of oxygen".
That did it. The class fell silent, busy trying to remember what they could about this. Can you help them out and draw it for them.
Answers available here.
Reversible binding of phosphate and control of conformation
Reversible binding of phosphate and control of conformation
Phosphorylation
Phosphorylation
The addition or removal of phosphate can cause reversible conformational change in proteins.
This is a common form of post-translational modification.
The activity of many cellular proteins, e.g. enzymes and receptors, is regulated in this way.
Task 38
Task 38
In your notes, sketch a phosphate group.
Can you think what R groups are likely to be targeted for phosphorylation?
The 20 amino acids are shown left. Phosphorylation requires an R group with a hydroxyl present.
This include:
Serine
Threonine
Tyrosine
All of these amino acid R groups can be phosphorylated by a group of enzymes called kinases.
In many cases, a kinase with use ATP as the source of phosphate.
This reaction is demonstrated in the image below.
The amino acid surrounded by the blue box is also serine - for the non-chemist, it is more recognisable and more in line with what we need to know at AH Biology level.
Task 39
Task 39
Include the structures of amino acids with R groups that are susceptible to phosphorylation by kinases.
Kinases and phosphatases
Kinases and phosphatases
Protein kinases catalyse the transfer of a phosphate group to other proteins. The terminal phosphate of ATP is transferred to specific R groups.
Protein phosphatases catalyse the reverse reaction.
Time for one of Mrs Kennedy's summary videos before looking at some past paper questions. Grab a ☕ and listen carefully.
Task 40
Task 40
Include a diagram in your notes to show the reversible nature of protein phosphorylation.
PPQ10
PPQ10
2023, Section 2, Qu3b
PPQ11
PPQ11
2023, Section 2, Qu3c
PPQ12
PPQ12
2018, Section 1, Qu6
PPQ13
PPQ13
2017, Section 1, Qu5
Phosphorylation brings about conformation change
Phosphorylation brings about conformation change
Phosphorylation brings about conformational change, which can affect a protein's activity. The activity of many cellular proteins, e.g. enzymes and receptors, is regulated in this way.
In the image above, you can see the conformational shift that occurs following phosphorylation.
Myosin phosphorylation
Myosin phosphorylation
Conformational change following phosphorylation of myosin within sarcomeres is the molecular basis of muscle contraction.
Some proteins are activated by phosphorylation while others are inhibited.
Adding a phosphate group adds negative charges. Ionic interactions in the unphosphorylated protein can be disrupted and new ones created.
In the diagram to the left, you can see the conformational shift within the protein that takes place. You can imagine that if the "site of activating phosphorylation" was around an active site, disruption of tertiary-level interactions might change the accessibility (for good or bad) of the active site for a substrate.
Watch the video link below by clicking on the pink box to hear an excellent summary of phosphoryation. This is such an important area that we will come back to it again during the Cell Signalling part of Topic 1.
Now go to SCHOLAR "2.5 Reversible binding of phosphate an the control of conformation" for consolidation.
Indeed, phosphorylation is such a hugely important part of cell communication that whole buildings are devoted to understanding the process more. The University of Dundee is home to the world-renowned Medical Research Council (MRC)-funded "Protein Phosphorylation and Ubiquitylation Unit". Yet another example of Scotland leading the way in cutting-edge Scientific research.
Task 41
Task 41
Using the image and link below (Crash Course: Muscle Cells), explain how phosphorylation facilitates muscle contraction.
Now go to SCHOLAR:
2.6 Learning Points
2.7 Extended resonse
2.8 End of topic test
to review and test your knowledge of Key Area 2.
Retrieval Practice
Retrieval Practice
Now is a good time to reflect on your most recent learning. With your notes closed, complete a set of Retrieval Cards for the key words displayed in the "image carousel" (left).
Like flashcards, write the key-term on one side and then the definition on the other.
Unlike flashcards, you should not look at your notes for the definition before you write it. Assess your own level of understanding first - how confident are you? Put a star of those cards you can complete without your notes and then write the definition; remember to check to make sure you are correct.
For those you are not sure of, you can look these up - but add a question mark to the front of these so you can reflect more heavily on these cards as you continue to revise throughout the year.
Your teacher might now issue you with Learner Check 8 to check your learning of the Topic 1 (Key Area 2) content so far.
Your teacher might now issue you with Learner Check 8 to check your learning of the Topic 1 (Key Area 2) content so far.
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