C. PROTEIN CHEMISTRY 2

Question 1

ways to determine protein structure

Answer 1

X-ray crystallography
NMR spectroscopy
Cryo-electron microscopy
AlphaFold

Question 2

where is animal insulin derived from

Answer 2

cows and pigs

Question 3

what type of amino acids does evolution conserve

Answer 3

those that are important to a protein’s structure and function across species
(compare sequences through aligning multiple ‘homologue’ sequences of a particular protein in different species)

Question 4

is insulin highly conserved

Answer 4

YES
Porcine and human insulin only differ in a single amino acid and bovine insulin varies by three amino acid

Question 5

what covalent forces are present in insulin

Answer 5

peptide bonds
disulphide bonds

Question 6

what non-covalent bonds are present in insulin

Answer 6

hydrogen bonds
Van Der Waals forces/interactions
hydrophobic interactions
electrostatic interactions
(ionic interactions and salt bridges)

Question 7

can peptide bonds be broken down

Answer 7

yes by hydrolysis but in very harsh (they are every stable) chemical conditions under 6M acid/alkali or by proteases under physiological conditions hence why storage is important

Question 8

can disulphide bridges be broken down

Answer 8

yes by reduction with β-mercaptoethanol (reducing agent containing thiols) to re-form cysteines

Question 9

why do hydrogen bonds contribute most to stability in proteins

Answer 9

they are furthest away from water which would disrupt them

Question 10

how are hydrogen bonds disrupted

Answer 10

heat or high salt solutions which cause heat

Question 11

what is optimum orientation of H-bonds

Answer 11

X-H points directly to the lone pairs so that the angle between X, H and Y is 180 degrees

Question 12

are VDW forces weak

Answer 12

yes but there are many of them and they are short dipole-dipole (δ+ & δ-) interactions between close atoms

Question 13

how are VDW forces disrupted

Answer 13

heat or denaturing agents like detergents, high salt solutions

Question 14

where does π-π overlap occur

Answer 14

between π electron clouds delocalised over rings and bonds (eg - aromatic rings)

Question 15

how are π-π overlaps disrupted

Answer 15

heat

Question 16

are electrostatic interactions strong

Answer 16

yes, inversely proportional to the distance between 2 charged groups

Question 17

how are electrostatic interactions broken

Answer 17

changes in pH or high ionic strength

Question 18

how do hydrophobic interactions occur

Answer 18

non-polar side chains of amino acids forced together in aqueous environments to minimise their disruptive effect on the hydrogen-bonding network of water molecules

Question 19

what is the distribution of amino acid residues in soluble proteins

Answer 19

charged/polar amino acids map to the surface (glutamic acid, serines, arginine) and non-polar are buried in the core (leucine, phenylalanine, proline)

Question 20

what is special about membrane proteins

Answer 20

they have extensive hydrophobic regions (ie the helices and sheets)

Question 21

what drives protein folding into 3D structure

Answer 21

non-polar side chains cluster in interior of protein so contact with water is avoided
polar side chains form hydrogen bonds with surrounding water molecules

Question 22

how do proteins fold to the native conformation

Answer 22

• go through intermediate states on their way to a stable low energy tertiary structure
• folding begins with formation of local segments of secondary structure. A so-called ‘molten globule’ can form by ‘hydrophobic collapse’ (all hydrophobic side-chains suddenly clump together), a structure in which the secondary structure elements of the protein are mostly formed
• chaperone proteins: assist in the proper folding of proteins in the cell

Question 23

example of protein that we eat which undergoes denaturation

Answer 23

eggs but re-naturation isn’t possible for eggs (is for others)

Question 24

how does denaturation occur

Answer 24

extreme changes in pH, temperature or addition of detergents

Question 25

structure of insulin in mature form

Answer 25

A chain - 21 residues
B chain - 30 residues
molecule linked 2 inter-chain and 1 intra-A-chain disulfide bridges
(C-chain and signal peptide are removed)

Question 26

how does insulin exist in dilute solutions like circulation

Answer 26

monomers (globular molecule) - can bind to receptor and are biologically active

Question 27

how does insulin exist in crystals and in β-cell secretory granules

Answer 27

hexamers
(at micromolar concentrations, insulin dimerises, and in the presence of zinc and iron, it further associates into hexamers)

Question 28

how are the biologically active monomers formed

Answer 28

Hexamers precipitate in crystalline form owing to the pH (around 5.5) and the presence of zinc and calcium ions

Upon extracellular release, the hexamers dissociate into dimers and eventually into monomers

Question 29

how does insulin form fibrils

Answer 29

occurs when the native state is destabilised, insulin monomer undergoes partial unfolding and the helical polypeptide hormone aggregates to form amyloid fibrils

complications:
when insulin is present at high concentrations ie - at the site of repeated insulin injection in diabetics and in clinical formulations, fibrillations can interfere with the requirement of equal amounts of functional insulin in each dose

Question 30

insulin surface features

Answer 30

monomer has 2 extensive non-polar surfaces
1. aromatic and buried upon dimer formation
2. buried upon hexamer formation

insulin uses the same surfaces for binding to its cognate receptor that it does for self-assembly

Question 31

when is rapid-acting forms of insulin used

Answer 31

meal times eg - Insulin lisper, insulin aspart, insulin glulisine

Question 32

how do rapid-acting insulin analogs work

Answer 32

after subcutaneous injection, the proportion that is bound in the form of dimers and hexamers is lower, which means that the monomeric form of the molecule can be absorbed
at the point of injection more quickly

Question 33

when is long-acting forms of insulin used

Answer 33

basal requirements

Question 34

how does insulin aspart work

Answer 34

proline 28 (neutral) in B-chain is substituted with an aspartic acid residue (-vely charged)
there is increased charge repulsion which prevents formation hexamers