Proteins

Question 1

What are the functions of proteins

Answer 1

defence, structure, catalysis, transport

Question 2

What is the basic structure of amino acids

Answer 2

linear polymer of amino acids which determines its 3D structure

Question 3

How can amino acids form a zwitterion

Answer 3

H from COOH lost to H2N making +H3N and COO-

Question 4

How does hydrogen bonding occur in amino acids

Answer 4

by the N having a lone pair and acting as a donor, essentially attracting and sharing H. C=O is a good H bond acceptor. Can form between different backbones of protein chains

Question 5

Describe the alpha helix structure

Answer 5

local regions of structure held together by hydrogen bonds between backbone chain. R groups are projected away from the centre of the helix. 3.6 residues a turn. Side chains project at 100 degrees to proceeding one. It brings together in space groups which are separated in the primary structure.

Question 6

What are helix formers

Answer 6

means these amino acids have a high propensity to cause alpha helix to form

Question 7

Helix destablisers

Answer 7

can find in alpha helix, but unlikely and if lots of them present tends to make it impossible for a helix to form

Question 8

Helix breaker

Answer 8

e.g., proline. Prevents formation of helix’s because of its structure the R group loops back and binds to amino ergo bond that would usually rotate, cannot anymore ergo impossible for chain to spiral as it cant bend at the correct point

Question 9

Describe beta pleated sheet structure

Answer 9

secondary structures are held together by backbone interaction, H bonds stablilise. very flexible laterally. Parallel beta sheet are sometimes less stable than antiparallel due to angles involved in H bond.

Question 10

Silk’s beta pleated sheet structure

Answer 10

cannot stretch in a longitudal direction because of interlocking links with beta strands, bond in the backbone have rotated to be at longest length, then locked in place and stabilised by H bonds meaning can’t stretch further

Question 11

Tertiary structure features

Answer 11

packing of secondary structures, sidechain interactions - some hydrophobic and some hydrophillic, van der waals forces, disulfide bridge

Question 12

What is the entropic effect

Answer 12

e.g., hydrophobic collapse. Hydrophobic molecule makes water molecules from a ‘cage’ (forces a structure, energy driver for chaos). Water molecules thus more ordered than if the hydrophobic molecule was not there. Entropic affect forces together lots of hydrophobic molecules into centre, producing a folding effect which is very strong. Different parts now interact affecting structure and function

Question 13

Quaternary structure

Answer 13

assembly of more than one polypeptide chain e.g., haemoglobin tetramer

Question 14

What is HIV proteinase

Answer 14

poly-protein processing. Essential for maturation and assembly to help HIV infect other cells

Question 15

Folding - function

Answer 15

structure/activity (what interacts with environment), necessary for drug design, necessary for protein engineering

Question 16

What is sodium dodecyl sulphate (SDS)

Answer 16

a detergent which unfolds proteins and coats unfolded chain

Question 17

What is PolyAcrylamide Gel electropheresis (PAGE)

Answer 17

inclusion of marker proteins of known molecular mass allows molecular mass of other proteins to be estimated

Question 18

Precipitation by ammonium sulphate (salting out) - purification

Answer 18

Dissolved salt also needs to be solvated so adding ammonium sulphate. To stay in solution a protein needs to be decreases the water available to solvate solvated by a “shell” of water molecules the protein

Assess supernatant & redissolved precipitate for presence of your protein

Depends on the polarity of the protein surface. This determines how soluble it is in water

Question 19

Isoelectric focusing - protein purification

Answer 19

Isoelectric point (pI): pH where a molecule has no net charge

Uses an immobilized pH gradient and an electric field

The sample containing mixture of proteins is applied

The proteins move either towards + or - depending on their net charge

As they move along the pH gradient their net charges are reduced

When they reach a pH at which their net charge is zero they stop This is the isoelectric point (pI)

The gel is stained with Coomassie Blue or silver nitrate

Question 20

Protein detection

Answer 20

most proteins are colourless, proteins absorb UV light at 280nm because of aromatic amino acid residue

Question 21

Seize exclusion chromatography (SEC) - gel filtration

Answer 21

Separation according to size (molecular sieving). Molecular architecture of bead - cross linked polydextran, large molecules cannot enter

Question 22

Determination of protein structure

Answer 22

SDS page gives monomer size, SEC gives native size, combination of SDS and SEC can determine the quaternary structure

Question 23

Ion exchange chromatography

Answer 23

Depends on ionisable groups in protein (so pH is important). Interaction of charged groups on protein with charged matrix in column. Polymer substituted with charged group.
Cation exchange –ve matrix interacts with cations: +ve charges on protein

Anion exchange uses different, +ve charged matrix to interact with –ve charges on protein

Question 24

Affinity chromatography

Answer 24

Some proteins have selective affinity for a particular structure e.g., enzyme and substrate. Specific affinity can be exploited, often with spectacular success for purification