Proteins

Question 1

Describe the general structure of an amino acid

Answer 1

Tetrahedral and chiral molecule
alpha-carbon in the centre, bonded to an amine group (NH2 or NH3+), a carboxylic acid (COOH or COO-), a hydrogen and an R-group

Question 2

Which amino acid enantiomer is present in mammals, and where can the other be found?

Answer 2

L-amino acid
The D-amino acid can be found in the peptidoglycan bacterial cell wall and mimicked in some antibiotics to interfere with bacterial metabolism

Question 3

What are the different side chain groupings for amino acids? Give examples for each.

Answer 3

Positively charged: arginine, lysine, histidine (can be uncharged)

Negatively charged: aspartate/aspartic acid, glutamate/glutamic acid

Polar uncharged: asparagine, glutamine, serine, threonine

Hydrophobic aromatic: phenylalanine, tyrosine, tryptophan

Hydrophobic aliphatic: glycine, alanine, valine, leucine, isoleucine, methionine, cysteine, *proline

*Structural: proline, because its aliphatic side chain bonds back onto the amine side group, and so it creates a kink in the pp chain

Question 4

Which is the only amino acid that cannot form stereoisomers and why?

Answer 4

Glycine, as its R-group is just a hydrogen, and so, due to the presence of two of the same side groups, the molecule is symmetric and superimposable on its mirror image

Question 5

Describe the peptide bond and its formation

Answer 5

Formed by condensation reaction
Between alpha-amine and alpha-carboxylic acid groups of different amino acids
Planar and rigid, as the bond exhibits partial double bond character
Almost always in trans configuration, where hydrogen of the NH is on opposite side of double bond to the oxygen on CO

Question 6

How does the polypeptide chain form 3D shapes if the peptide bond is rigid?

Answer 6

Free rotation around the alpha-C to N and C atoms respectively

Question 7

Explain the stability and structure of alpha helices

Answer 7

Most stable helical secondary structural arrangement as there is minimal strain on the inter-amino acid hydrogen bonding
R groups point outwards from helix
Hydrogen bond formed between every 4 amino acids, between the carbonyl O and the amino H
Periodicity of 3.6 residues

Question 8

Explain the stability, structure and characteristics of beta sheets

Answer 8

Fully extended pp chain
Adjacent chains can run parallel or antiparallel
Planarity causes protrusion of R groups above and below the sheets
Hydrogen bonds formed between the carbonyl O and amino H of aligned sheets
Strong and rigid

Question 9

Explain the stability, structure and function of loop regions

Answer 9

Stabilized by hydrogen bonding between carbonyl O of residue 1 and amino H of residue 4 on the other side of the turn
The second and third residues are often proline (due to lack of amino H for hydrogen bonding) or glycine, as they can’t contribute any stabilizing bonds

Question 10

Define primary, secondary, tertiary and quaternary structure

Answer 10

P - the linear sequence of amino acids in the pp chain
S - the local interactions and folding of the amino acid chain
T - the overall structures formed and spatial arrangement of amino acids in the pp chain
Q - the complex formed by interactions between multiple polypeptide chains

Question 11

What is a domain?

Answer 11

A specific region of a polypeptide chain which has an independent function (e.g. ATP-binding site, zinc finger, immunoglobulin fold etc.) that may be common to other polypeptide chains with an overall different tertiary structure

Question 12

What reaction forms a disulphide bridge? Between which two residues can it be formed, and what residue does it generate?

Answer 12

Oxidation (SH SH –> S-S + 2H+)

Occurs between the R groups of two cysteine residues, and forms one cystine residue

Question 13

What is the difference between a homo-oligomeric protein and a hetero-oligomeric multi-subunit protein?

Answer 13

Both have quaternary structure, and are thus complexes of multiple pp chains, however homo-O contains same pp subunits whereas hetero-O contains different pp subunits.

Question 14

What is cooperativity? Give an example of it in action

Answer 14

When an enzyme has more than one active site, and the binding of one substrate to an active site will influence the binding of another substrate to another active site.
In haemoglobin, the O2 affinity curve is sigmoidal because the binding of the first O2 to one of the haem groups causes an allosteric change in the protein making binding to the other binding sites easier, and so on.

Question 15

What is an allosteric change?

Answer 15

A conformational change occurring in an enzyme that influences the shape of one binding site due to the binding of a ligand at another, distant binding site

Question 16

Describe the post-translational modifications below:

1. Disulphide bonding
2. Crosslinking
3. Peptidolysis
4. Glycosylation
5. Phosphorylation
6. Adenylation
7. Farnesylation

Answer 16

1. Oxidation reaction removing 2H+ from two cysteine residues to form an S-S covalent bond, creating a new, single cystine residue
2. Covalent crosslinks between molecules
3. Removal of parts of the protein after synthesis
4. Addition of carb groups
5. Addition of phosphate groups to specific residues
6. Addition of adenosine monophosphate
7. Attachment of an unsaturated C15 hydrocarbon group called a farnesyl anchor which can insert into the plasma membrane

Question 17

What general roles can post translational modifications have, using an example for each?

Answer 17

Regulation - phosphorylation by PKA, for example, can turn a protein on or off

Targeting - glycosylation, for example, can act as a signal to intracellular targeting or sorting machinery

Turnover - labelling with ubiquitin protein, for example, can mark proteins out for degradation by proteosomes

Structural - crosslinking between collagen molecules, for example, can greatly increase the strength of the resulting fibril

Question 18

What is collagen, where is it found and what is the structure of collagen type 1?

Answer 18

Structural, fibrous protein
Found in tendons, ligaments and other connective tissues
A triple helix of two alpha 1 chains and one alpha 2 chain

Question 19

Describe the collagen polypeptide sequence and the helix/triple helix it forms

Answer 19

A glycine occurs every third residue as it is the only residue small enough that it can be positioned at the centre of the triple helix
There are repeats of the sequence -gly-X-Y, with X usually being pro and Y usually being OHpro
The helix of each chain is left handed, but the triple helix itself is right handed
Two amino acids, OHpro and OHlys, are formed post-translationally and are a key feature of the molecule

Question 20

Outline the stabilizing features of tropocollagen

Answer 20

Interchain hydrogen bonding between peptidyl amino and carbonyl groups of the gly, and the hydroxyl groups of the OHpro

Question 21

What are the different stages in collagen fibril formation, and note where each stage occurs?

Answer 21

Pre-procollagen = individual chains (nucleus to RER)
Procollagen = triple helix formed, but with C and N terminal propeptides (RER to Golgi)
Tropocollagen = only the triple helix, terminal propeptides cleaved (extracellular)
Collagen fibril = tropocollagen molecules align parallel in a staggered arrangement, covalently crosslinked via lys and OHlys residues’ side chains (extracellular)

Question 22

What are the two causes, and symptoms, of Ehler-Danlos syndrome?

Answer 22

Causes: Defective enzyme that cross links hydroxylysine residues, and defective enzymes that cleave terminal propeptides of procollagen to form tropocollagen

Symptoms: Hyperextensible skin, recurrent joint dislocation

Question 23

What are the two causes of osteogenesis imperfecta? What are the symptoms?

Answer 23

Broadly speaking, OI is caused by an inability to form the triple helix. This can occur due to gene defects producing an abnormally short alpha 1 chain which cannot form a stable helix and therefore fibre, or the substitution of gly for arg or cys which are two big, and therefore destabilise the centre of the helix

Symptoms: brittle bones, repeated fractures, bone deformities in infancy

Question 24

What is the function of histone proteins?

Answer 24

To package DNA in order to make it more space efficient, but also to regulate gene expression

Question 25

What properties of histones allow them to associate permanently with DNA?

Answer 25

They are globular and have a cationic surface which can neutralise the negatively charged phosphates of the DNA backbone

Question 26

How do histones arrange to form nucleosomes?

Answer 26

They form octamers, made from two layers of the four histones H2A, H2B, H3 and H4, around which 146 base pairs of DNA wind. The nucleosomes are stabilised by H1, which controls the length of the linker DNA and prevents unwinding

Question 27

What is the name of the helical array that nucleosomes of DNA can form?

Answer 27

Solenoids

Question 28

What is hydrolysis?

Answer 28

The splitting of a bond using water (e.g. breaking a peptide or glycosylic bond)

Question 29

What is the reverse of hydrolysis?

Answer 29

Condensation, where water is formed

Question 30

What is group transfer?

Answer 30

The moving of one biochemical group from one molecule to covalently bond to another

Question 31

What is a protein subunit, and hence a multimeric protein?

Answer 31

A protein subunit is a single pp chain that can assemble with other pp chains to form a multimeric protein

Question 32

Explain PKA’s regulation, and give it a name.

Answer 32

PKA is a heteromeric enzyme whose functional subunits are only activated when the enzyme undergoes a conformational change. This only occurs when cAMP binds to its regulatory subunits to activate its catalytic subunit.
Allosteric control

Question 33

Define isozyme, and give an example

Answer 33

An enzyme that exists in multiple different isoforms in different tissues of the body
Lactose dehydrogenase

Question 34

What is a multienzyme complex, and give an example?

Answer 34

A complex formed by the association of multiple individual enzymes to carry out a certain function together
Pyruvate dehydrogenase is formed of E1-E3 ubiquitin ligases- E1 has a tetrameric structure containing the active sites for NAD+ and pyruvate, but these molecules are held in place by the other enzymes of the multienzyme complex so they can react

Question 35

Define:

1. Michaelis constant
2. Maximal velocity
3. Turnover number
4. Specificity constant
5. Ki or inhibitor constant

Answer 35

1. Substrate conc. at which the the reaction rate is half Vmax; the lower Km, the higher the enzyme’s affinity for the substrate
2. Reaction rate at which only limiting factor is enzyme concentration
3. Number of substrate molecules that can be turned into product when enzyme is saturated with substrate
4. The efficiency with which the forwards reaction is catalysed
5. Concentration of inhibitor required to half the Vmax when the enzyme is otherwise saturated with substrate

Question 36

Why does myoglobin have a higher affinity than haemoglobin for their shared substrate?

Answer 36

The haem group in myoglobin is in more of a relaxed state than those of haemoglobin, which are in more of a tense state, and the relaxed state promotes higher enzymatic activity as it exposes the active site more.

Question 37

Give an example of a clinically relevant enzyme inhibitor (commercial and functional name)

Answer 37

Mevinolin/Statin, a HMG-CoA Reductase inhibitor
HMG-CoA reductase is an important enzyme in the synthesis of mevalonate, a precursor to cholesterol in the cholesterol synthesis pathway
Mevinolin is a potent fungus-derived competitive inhibitor of the reductase enzyme