Session 5.1a - Group Work

Question 1

Medical Cell Biology and Genetics

Session 5

Interactive

During the worksession there will be some molecular models available for you to use to examine the structure of amino acids. In your group use 1 MolyMod kit to make 1-2 amino acids. Have a go at joining these together with a peptide bond and look at the effect of having a cis peptide bond and some of the consequences of rotating the bonds on either side of the peptide bond. Try joining them together with amino acids made by other groups.

Answer 1

Interactive

Question 2

1. The structures of the side chains of all 20 amino acids are shown below (shaded). Using your knowledge of chemistry, for each amino acid classify them using one or more of the following terms:

Hydrophobic
Hydrophilic
Polar
Non-polar
Basic
Acidic
Aliphatic
Aromatic

Answer 2

Hydrophobic - Gly Ala Val Leu Ile Met Trp Phe Pro
Hydrophilic - Asp Glu Lys Arg His Ser Thr Cys Tyr Asn Gln
Polar - Asp Glu Lys Arg His Ser Thr Cys Tyr Asn Gln
Non-polar - Gly Ala Val Leu Ile Met Trp Phe Pro
Basic - Lys Arg His
Acidic - Asp Glu
Aliphatic - Gly Ala Val Leu Ile Met Pro
Aromatic - Trp Phe Tyr

Question 3

2. Explain why some amino acid side chains are charged at physiological pH.

Answer 3

All amino acids have a carboxyl group and an amino group, which are dissociated at physiologic pH. However, because these cancel each other out in solution (zwitterions) - not all amino acids are charged. Therefore, it is the SIDE CHAINS of amino acids which determine their chemical properties.

The side chains of some amino acids have ionisable groups - these include the carboxyl group and amino group. These can either proton donate or proton accept, respectively. At physiological pH, the carboxyl group is dissociated, giving the carboxylate ion (COO-), whilst the amino group is protonated (NH3+), thus, those amino acids with these groups in their side chains are charged at physiological pH.

Question 4

3. What does the pKa value of an amino acid side chain tell you about that chemical group?

Answer 4

The pKa value is the pH at which there is no overall net charge on that acid or base.

It also tells you how strong/weak that chemical group is as acting as an acid/base:

The smaller the pKa, the stronger the acid/weaker the base
The larger the pKa, the stronger the base/weaker the acid

So glutamic acid would have a small pKa, whereas lysine would have a large pKa.

It also tells us at which pH the side chain can act as a buffer (resist a change in pH) by acting as a weak acid/base - maximal buffering occurs when pH = pKa but buffering can still occur +/-1 either side of this value.

By understanding that pH = pKa we can remember that the lower the pH value the more acidic a solution/group etc. is, thus the stronger the acid at a lower pKa; and the higher a pH value the more alkaline a solution/group etc. is, thus the stronger the base at a higher pKa.

pKa = -log Ka (acid dissociation constant)
Ka = [H+][A-]/[HA}

So the larger the Ka, the stronger the ACID, because most of the HA has dissociated into H+ and A−. Conversely, the smaller the Ka, the stronger the BASE (weaker the acid) because less HA has dissociated.

Question 5

4. The side chain of aspartic acid has a pKa value of 2.8. What does this mean?

Answer 5

It is a very strong acid because it has a low pKa value, and is therefore a very good proton donor (willingly gives away its proton)

At physiological pH (7.4) it will be deprotonated.

Question 6

5. The side chain of the amino acid histidine can exist in two different ionized forms as shown below:

The pKa for the equilibrium between the protonated and deprotonated forms of histidine (pkR) is approximately 6.0.

a) Which is the predominant form of histidine at pH7.0?

Answer 6

What should you be thinking?

At a pKa of 6 there will be equal amounts of the protonated form (on the left) and the deprotonated form (on the right)

If we add more H+ ion (DECREASE the pH) the equilibrium shifts to the left and the PROTONATED form predominates

If we take away H+ ion (INCREASE the pH) or add OH- equilibrium shifts to the right and the DEPROTONATED form predominates

[pH 7.0 > pK 6.0
Therefore the deprotonated form of histidine will predominate.]

Question 7

5. The side chain of the amino acid histidine can exist in two different ionized forms as shown below:

The pKa for the equilibrium between the protonated and deprotonated forms of histidine (pkR) is approximately 6.0.

b) Calculate the ratio of protonated to deprotonated forms of the side chain at pH7.0.

Need to use the Henderson-Hasselbalch equation to calculate this:

Answer 7

pH = pK + log [base/[acid] OR pH = pK + log [deprotonated] / [protonated]

7. 0 = 6.0 + log [deprotonated] / [protonated]
8. 0 = log [deprotonated] / [protonated]

10^1 = [deprotonated] / [protonated]

THEREFORE, DEPROTONATED : PROTONATED = 10:1

Question 8

6. (a) What is meant by the isoelectric point (pI) of a protein?

Answer 8

The isoelectric point (pI) is the pH at which a COMPOUND (e.g. protein, amino acid) is electrically neutral (has no overall net charge) - i.e., the sum of the positive charges equals the sum of the negative charges.

Question 9

6. (b) The protein serum albumin has an isoelectric point of 5.0. If this protein was placed in an electric field at physiological pH, would it move towards the positive or negative electrode? Explain your answer.

Answer 9

An isoelectric point of 5.0 means that the protein has a lot of acidic amino acid residues (pI < 7). At physiologic pH (7.4), this protein would be largely deprotonated (negatively charged). Proteins migrate towards the oppositely charged electrode, thus, the protein would move towards the positive electrode in an electric field.

Question 10

6. The protein serum albumin has an isoelectric point of 5.0. If this protein was placed in an electric field at physiological pH, would it move towards the positive or negative electrode? Explain your answer. [ANSWERED]

(c) Which direction would the same protein migrate at:
i) pH 5.0

Answer 10

The isoelectric point is the pH at which there is no net charge. Thus, at pH 5.0, there is no net charge and the protein would be attracted to either electrode.

Question 11

6. The protein serum albumin has an isoelectric point of 5.0. If this protein was placed in an electric field at physiological pH, would it move towards the positive or negative electrode? Explain your answer. [ANSWERED]

(c) Which direction would the same protein migrate at:
ii) pH 4.0

Answer 11

At pH 4.0, the pH is lower than the pI so more of the protein will be positively charged. Thus, it is more likely to migrate towards the negative electrode.

Question 12

7. Histones are proteins abundant in eukaryotic cell nuclei where they form part of chromatin. The isoelectric point of histones is very high, about 10.8.
   (a) What charge will histones have under physiological conditions?

Answer 12

Physiological conditions = pH of 7.4

The pI of histones is high, which means it contains a lot of basic amino acid residues. As 10.8 is much higher than 7.4, it will be positively charged under physiological conditions.

Question 13

7. Histones are proteins abundant in eukaryotic cell nuclei where they form part of chromatin. The isoelectric point of histones is very high, about 10.8.
   b) Which type of amino acids must be present in large numbers in these proteins?

Answer 13

Basic amino acids: lysine, arginine and histidine

Question 14

7. Histones are proteins abundant in eukaryotic cell nuclei where they form part of chromatin. The isoelectric point of histones is very high, about 10.8.
   c) Why do you think that histones have this charge?

Answer 14

Histones are a family of basic proteins that associate with DNA in the nucleus and help condense it into chromatin. Nuclear DNA does not appear in free linear strands; it is highly condensed and wrapped around histones in order to fit inside of the nucleus and take part in the formation of chromosomes.

Histones are basic proteins, and their positive charges allow them to associate with DNA, which is negatively charged.

Question 15

8. Many genetic diseases are caused by changing a single amino acid in a protein. However, the severity of the disease can depend on the nature of the change. Using your knowledge of amino acid characteristics explain whether each of the following changes is likely to have a large or small effect on protein function. In thinking about this question you should bear in mind that amino acids have different chemical properties as well as differences in size of the R group.
   (a) Arginine changed to Lysine

Answer 15

Arginine = basic amino acid
Lysine = basic amino acid

Both arginine and lysine exhibit the same chemical properties, as they are both basic amino acids (albeit arginine more so). Thus, a mutation of the two will not cause too much difference as the chemical bonding will still react in the same way.

Arginine is also only very slightly bigger than lysine - thus again, there will be not much change due to the physical properties of the two faring quite similarly.

A mutation from arginine to lysine would therefore have little effect on the protein (arginine is bigger and more basic but they are similar).

Mutations such as this are termed conservative replacements.

Question 16

8. Many genetic diseases are caused by changing a single amino acid in a protein. However, the severity of the disease can depend on the nature of the change. Using your knowledge of amino acid characteristics explain whether each of the following changes is likely to have a large or small effect on protein function. In thinking about this question you should bear in mind that amino acids have different chemical properties as well as differences in size of the R group.
   b) Arginine changed to Glutamate

Answer 16

Arginine = basic amino acid
Glutamate = acidic amino acid

An arginine to glutamate mutation would have large ramifications for the protein, as arginine is a basic amino acid and glutamate is an acidic amino acid so their bonding properties are completely opposite. Glutamate would not only not wish to bind to the amino acids the original arginine was proposed to (repel them), it would also try to bind excess amino acids that arginine would not. Thus, the whole electrochemical properties would be out of whack, and the pI of the protein would also be changed.

Even if the two were in the same chemical group, glutamate is quite a bit more truncated than arginine, so there would be physical differences if not for the chemical ones.

Therefore, there would be a large effect on protein function. If this was an enzyme, and you often find charged residues in the active site of an enzyme which interact with the substrate, it might affect the Km of the enzyme.

Question 17

8. Many genetic diseases are caused by changing a single amino acid in a protein. However, the severity of the disease can depend on the nature of the change. Using your knowledge of amino acid characteristics explain whether each of the following changes is likely to have a large or small effect on protein function. In thinking about this question you should bear in mind that amino acids have different chemical properties as well as differences in size of the R group.
   c) Alanine changed to Glycine

Answer 17

Alanine = aliphatic hydrophobic compound
Glycine = aliphatic hydrophobic compound

An alanine to glycine mutation would not present with too many problems as they are in the same chemical group - they are both aliphatic hydrophobic compounds. Importantly, they are also of very similar size - alanine is larger by a single CH2. Thus, there would be little change in protein function (although it is to be noted that glycine is the smallest amino acid, and it could be that alanine is just too big to fit into whichever gap is proposed for glycine. However, in general, there will be little effect.).

Question 18

8. Many genetic diseases are caused by changing a single amino acid in a protein. However, the severity of the disease can depend on the nature of the change. Using your knowledge of amino acid characteristics explain whether each of the following changes is likely to have a large or small effect on protein function. In thinking about this question you should bear in mind that amino acids have different chemical properties as well as differences in size of the R group.
   d) Alanine changed to Tryptophan

Answer 18

Alanine = small hydrophobic aliphatic amino acid
Tryptophan = large hydrophobic aromatic amino acid

Although alanine and tryptophan both comprise 2 of the several hydrophobic amino acids, they exhibit a vast difference in shape and size. Alanine is a very small aliphatic amino acid with a singular CH3 side chain, whereas tryptophan is a large amino acid that has an indole side chain, making it aromatic. Not only is this much larger than alanine, it also has a completely different shape (because it is no longer straight).

Thus, although the chemical binding properties will be similar, and bonding properties will not be altered too greatly, there might be a medium effect on protein function, as the shape of tryptophan is completely different, so the protein won’t be able to pack into the same shape as it was before. Depending on the function of the protein (requires it to fit into a little space) and if this is in a key area (e.g., the active site of an enzyme) this may or may not have a large effect on protein function.

Question 19

[Slide]
Review Session
Session 5 Question 1

Look at the structure of aspartate.

What should you be thinking?
What types of interaction can it make?

Answer 19

What should you be thinking?
Charged - negative charge
Acidic amino acid residue - donated proton
Polar

What types of interaction can it make?
Ionic interactions - -ve charge
H-bonds - via carbonyl oxygen

Question 20

Review [Speech] Session 5 Question 1

What do we need to know about amino acids? Use aspartate as an example.

[Explanation not notes]

Answer 20

Think about the structure of the AAs but also particularly their side chains, but what they actually mean.

Aspartate – thinking about the side chain, this bit here (bg of purple) – remember when talking about proteins only bit of AA we’re interested in is amino acid residue – the side chain - the rest of this has gone – formed our peptide bonds.

What should you be thinking?
• negatively charged. Carboxylate anion – (was COOH, now COO-) so aspartate.
• acidic amino acid residue.
Note: what is our definition of an acid – proton donor? Can aspartate donate a proton, no, it’s already donated a proton – if it was the proton donor it would be called aspartic acid., now aspartate – the anion. So acidic AA has already donated its proton - -ve charged. So acidic AAR are negative charged.
• Polar – diff from this end to this end - -ve charged (COO-) more neutral (CH2).

What types of interaction can it make?
• Ionic interactions – can be interacting with +ve charged AARs in the protein – e.g. Lys, Arg.
• H-bonds – from C=O. (It could also make hydrophobic interactions if it wanted to (CH2 side))

Question 21

[Slide]
Review Session 5 Question 1

Do we expect you to know the structure of all the amino acid side chains and to remember their properties

Answer 21

“No!! But you should be able to define the properties if we give you the structure.”

It’s really important that we DON’T expect you to know the structure of all the AAs. Need to know basic structure of an AA, but NEVER see an exam q that says draw the structure of aspartate. Might GIVE it to you and ask you to classify it – and say okay it’s been mutated to arginine – why might effect be of changing it to arginine? -ve charged changed to +ve charged, maybe bc in active site of enzyme, maybe interaction with substrate – often find charged residues in active sites that interact with substrate – if -ve charged residue interacting with +ve charged residue that’s fine. But change it to +ve charged, will it still interact? No, it will actually repel it, so it might actually affect Km of the enzyme. Not asking for factual recall but asking you to apply your knowledge. So want you to use the structure of an AAR we will GIVE you the structure.

Question 22

[Review Session 5 Question 1]

Is histidine aromatic or aliphatic?

Answer 22

(actually missing a CH2 group between a and gamma carbons in worksheet image, but doesn’t make any difference for us BECAUSE WE DON’T NEED TO KNOW THE STRUCTURE)

NEITHER.

Aliphatic = chain of carbons and hydrogens e.g.leucine, but His also has N in.

Aromatic = benzene-like ring. His has an imidazole ring.

Question 23

[Review Session 5 Question 5a]

Explain your answer.

Answer 23

Important to think about it as a fairly conceptual level.

So histidine is going to exist in 2 diff forms: +ve charged or protonated form, and in neutral or deprotonated form.

It has a pKa value – log of acid dissociation constant, and this tells you how likely this is to disassociate. So the pKa (pKr means the pKa value of the side chain, not the amino group or carboxyl group or thing as a whole) of 6 means at pH of 6 there’s 1:1 ratio - eqm.

What would happen if we add more H ions? i.e. drop pH to pH 5. Well should draw in another H ion on right side to show eqm. What happens if we add more H ions, H ions combined to right form to drive eqm towards left, trying to shift it to pH 6 - eqm change to resist the change.

Similarly, if we take H ions away from right side, the eqm will react by moving to the right to release more H ions.

So know this concept

Question 24

[Review Session 5 Question 5b]

Explain your answer.

Answer 24

(HH equation - highly unlikely we’d ask you a q relating to this on MCBG but you might come across it in Resp later on related to acid/base eqm)

Acid (proton donor) = protonated (left)
Base (proton acceptor) = deprotonated (right)

Anti-logs
Remember log is the power we raise 10 to get that number. So anti-log is 10^1 in this case.

So that tells you there’s a ratio of 10:1 of deprotonated to protonated, which fits in with what you said before.

So if you come across a calculation like this think about it and think whether it makes sense – follow it through logically to decide whether you’ve got to a point that makes sense.

Question 25

[Student question[

What do we need to know about the Michaelis-Menten equation?

Answer 25

Do we have to use it and memorise it? NO.

But we do need to know about the TWO important CONSTANTS: Km and Vmax - we’re interested in those bc they tell us something about the enzyme.

Review Session of Enzymes Session 6.