Proteins Flashcards
What are the functions of proteins
defence, structure, catalysis, transport
What is the basic structure of amino acids
linear polymer of amino acids which determines its 3D structure
How can amino acids form a zwitterion
H from COOH lost to H2N making +H3N and COO-
How does hydrogen bonding occur in amino acids
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
Describe the alpha helix structure
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.
What are helix formers
means these amino acids have a high propensity to cause alpha helix to form
Helix destablisers
can find in alpha helix, but unlikely and if lots of them present tends to make it impossible for a helix to form
Helix breaker
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
Describe beta pleated sheet structure
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.
Silk’s beta pleated sheet structure
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
Tertiary structure features
packing of secondary structures, sidechain interactions - some hydrophobic and some hydrophillic, van der waals forces, disulfide bridge
What is the entropic effect
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
Quaternary structure
assembly of more than one polypeptide chain e.g., haemoglobin tetramer
What is HIV proteinase
poly-protein processing. Essential for maturation and assembly to help HIV infect other cells
Folding - function
structure/activity (what interacts with environment), necessary for drug design, necessary for protein engineering
What is sodium dodecyl sulphate (SDS)
a detergent which unfolds proteins and coats unfolded chain
What is PolyAcrylamide Gel electropheresis (PAGE)
inclusion of marker proteins of known molecular mass allows molecular mass of other proteins to be estimated
Precipitation by ammonium sulphate (salting out) - purification
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
Isoelectric focusing - protein purification
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
Protein detection
most proteins are colourless, proteins absorb UV light at 280nm because of aromatic amino acid residue
Seize exclusion chromatography (SEC) - gel filtration
Separation according to size (molecular sieving). Molecular architecture of bead - cross linked polydextran, large molecules cannot enter
Determination of protein structure
SDS page gives monomer size, SEC gives native size, combination of SDS and SEC can determine the quaternary structure
Ion exchange chromatography
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
Affinity chromatography
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
