Protein tutorial

Based on Lectures 1-to-6 of BS125 (Proteins, Genes and Genetics).

This tutorial is to help you consolidate your understanding of material covered in the recent Proteins lectures (Lectures 1-to-6 of BS125 Proteins, Genes and Genetics). The questions require a mixture of factual recall and a more analytical approach using knowledge obtained from the lectures. Complete the worksheet and bring it with you to the tutorial on Friday Week 4. Do it from memory as much as possible and then use your lecture notes and textbooks to finish it if necessary.

1.

List, with examples, seven different functions of proteins.

Enzymes e.g. ATPase Defence e.g. Immunoglobulin A (in upper respiratory tract) Structural Proteins e.g. Collagen Transport Proteins e.g. Porins in cell membranes Hormones e.g. Insulin Contractile Proteins e.g. Actin in muscles Gene control e.g. transcription factors 2. Define the term electronegativity and arrange the following atoms in order of increasing electronegativity. Carbon, Oxygen, Nitrogen, and Hydrogen. Electronegativity is the ability of an atom to attract a bonding pair of electrons from a covalent bond Hydrogen, Carbon, Nitrogen Oxygen

3. Describe the four types of noncovalent interactions in biological molecules. Van der Waals forces formed when electrons (which are constantly moving) end up more to one side of the atom than the other. This creates an uneven distribution of charge, and the atom is now said to have a dipole. This then induces a dipole in neighbouring atoms. The two atoms become attracted to each other due to the opposing charge. Electrostatic Interactions formed between oppositely charged ions Hydrogen bonds formed between an electropositive hydrogen atom (a hydrogen atom attached to an electronegative atom like Oxygen or Nitrogen) and an electronegative element with a lone pair such as oxygen Hydrophobic Interactions when non-polar substances form clusters in aqueous solution and exclude water molecules

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4. Indicate whether the following can act as hydrogen bond donors or hydrogen bond acceptors ?

Donor -C-H -O-H -N-H

5.

Acceptor

Which amino acids of K, E, L, C, W, S are associated with the following side chains?

Nonpolar aliphatic Nonpolar aromatic Basic Acidic Contains sulfur S

6.

L W K E C

Contains an hydroxyl group

Under appropriate conditions, the side chain of lysine (pK ~10.0) can form electrostatic interactions with the side chains of either glutamate (pK ~4.4) or tyrosine (pK ~10.0). Which interaction is more likely to occur in proteins inside human cells, and why?

Glutamate. As glutamate has a negatively charged side chain at physiological pH and lysine has a positively charged side chain at physiological pH these two will form electrostatic interactions.

7.

You suspect that the protonated form of the imidazole ring of the amino acid histidine participates in a particular enzyme-catalysed reaction. Given that the pK value of the imidazole ring is 6.5, calculate the percentage of enzyme molecules active at pH 7.5.

pH = pKa + log ([A-] / [HA]) 7.5 = 6.5 + log ([A-] / [HA]) 1 = log ([A-] / [HA]) 101 = 10 = ([A-] / [HA]) Protein tutorial Page 2

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8. Discuss the statement the strength of noncovalent interactions is that they are weak. Non-covalent interactions are weak and so can be broken much more easily than covalent bonds. This is important as many biological processes rely on the ability of structures to be able to change shape/break apart. E.g. the unzipping of DNA would not be possible if the bases on the two different stands were bonded using covalent bonds.

9.

The -keratin of human hair is an example of a fibrous protein with extensive -helical structure and many disulfide bonds. Silk fibroin is also a fibrous protein, but it consists primarily of pleated sheet structure. If you were to grab onto both ends of an -keratin polypeptide and pull, you would find it to be both extensible (it can be stretched to about twice its length in moist heat) and elastic (when you let it go, it will return to its normal length). In contrast, a fibroin polypeptide has essentially no extensibility, but it has great tensile strength. Explain these differences.

Alpha helices form less non-covalent interactions than beta-pleated sheets as beta pleated sheets have a larger surface area over which these interactions form. This means that structures made from beta sheets have a larger tensile strength than those made from alpha helices, and so cannot be so easily stretched.

10. Christian Anfinsen and colleagues performed many experiments into the folding of proteins using the enzyme ribonuclease A (RNaseA) as their model protein. In their classic experiment (described in Sela, White and Anfinsen 1957 Science 125, 691692) they denatured RNaseA using -mercaptoethanol (BME) and 8M urea. Removing the BME and urea by dialysis allowed most of the denatured protein to refold into the active conformation. In contrast, removing the BME but NOT the 8 M urea results in less than 1% of the protein becoming enzymatically active (Anfinsen described the protein as scrambled). Explain why this approach is less efficient than the experiment in which the BME and urea are removed simultaneously by dialysis. The urea disrupts hydrogen bonds and hydrophobic interactions and mercaptoethanol (BME), disrupts disulphide bridges. When both are removed by dialysis, the enzyme begins to refold of its own accord as there is nothing to stop the non-covalent interactions from reforming. However, when BME is removed but urea is still present, less than 1% of the protein reforms. This is because the urea still present is preventing hydrogen bonds and hydrophobic interactions from forming. Protein tutorial Page 4

Disulphide bridges can still form (hence the less than 1% reformation) but there are very few of these interactions in RNaseA the most important noncovalent interaction is hydrogen bonds. Therefore, as hydrogen bonds cannot form, the protein does not reform to adopt its original shape.

The scrambled RNaseA can be converted to the active enzyme by adding a low concentration of BME (much lower than the amount required to denature the protein) or by adding the enzyme protein disulfide isomerase. Which of these two methods is the faster at refolding the ribonuclease, and why? The enzyme protein disulphide isomerase is faster than using BME. This is because protein disulphide isomerase is actively catalysing the breaking and reforming of the disulphide bonds.

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11. The major hemoglobin present during the latter two-thirds of fetal life (HbF) is a tetramer of 2 2 in which the -chains are very similar to the -chains found in adult hemoglobin (HbA, 22). One significant difference is that His 143 in HbA is replaced by Ser in the -chain of HbF. Structural studies indicate that His 143 sits in the central cavity of the HbA molecule and plays an important role in binding 2,3-bisphosphoglycerate (BPG). Explain how the transfer of oxygen from a mother to her fetus is facilitated by this difference between their hemoglobin molecules. BPG lowers the oxygen affinity of haemoglobin, promoting release of oxygen. It is important that the haemoglobin molecules of the foetus do not contain His143 as otherwise BPG would attach to this and the foetal haemoglobin would not have a high affinity for oxygen. It is vital that the foetal haemoglobin have a high affinity for oxygen as they need to take it from the haemoglobin of the mother.

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