amino acids

Question 1

what is a zwitterion

Answer 1

a molecule with functional groups, of which at least one has a positive and one has a negative charge
therefore, net charge of the molecule is zero

Question 2

what is the isoelectric point?

Answer 2

the pH at which a particular molecules carries no net charge
the zwitterion exists at this pH
above/below - molecule has net charge

Question 3

how to calculate the isoelectric point

Answer 3

mean of the two pKa values for amino acids with 2 ionisable groups

Question 4

what is interesting about zwitterions

Answer 4

at isoelectric point, zwitterion exists as a dipolar ion (positive amino and negative carboxyl group)
therefore, the zwitterion can exist as either an acid or a base
these
these molecules are amphoteric (dual acid-base nature), they are called ampholytes

Question 5

what is a postsynthetic modification?

Answer 5

common residues already incorporated into a polypeptide are modified (e.g. add phosphoryl, methyl, acetyl or adenlyl groups)

Question 6

what is the beer-lambert’s law:

Answer 6

A = ecl
A = absorbance
e = extinction coefficient
c= concetration (M)
L =path length (cm)

Question 7

2 peaks of interest via the beer-lambert law:

Answer 7

220nm =photons absorbed by carbonyl group (from carboxylic acid or amide group)
280n - aromatic residues

Question 8

how to work out concentration of protein?

Answer 8

protein concentration = (absorbance at 280nm/ el) x sample dilution

Question 9

how are carbons in amino acids numbered?

Answer 9

highestg priority to carbon in carboxyl group

Question 10

hydrogen bonding in terms of polypeptides?

Answer 10

carbonyls - hydrogen bond acceptors
amino groups - hydrogen bond donors

Question 11

how are polypeptides named?

Answer 11

start name from amino group

Question 12

features of peptide bonds?

Answer 12

has partial double bond chatacter = geometric isomers form (cis or trans)

cis = amide nitrogen and carbonyl are on same side of peptide bond
trans = opposite ides of the peptide bond

steric repulsion - hinders formation of cis configuration

Question 13

what is the exception to the cis and trans configurations?

Answer 13

proline - both cis and trans have a steric clash so you get a mix of the two

Question 14

what are motifs?

Answer 14

a combination of secondary structures (e.g. helix-turn-helix)

Question 15

features of globular proteins.

Answer 15

compact, soluble, no symmetry
if there are more than 2 globular units, these are called domains

Question 16

example of globular protein?

Answer 16

myoglobin - carry oxygen in muscle tissue
interior - non polar (except His - which binds Fe and O2)
exterior - polar and non-polar

Question 17

what is the hydrophobic effect?
problem created by NH and CO?

Answer 17

forms water-soluble proteins (hydrophillic on outside) with non-polar cores
unpaired NH and CO are hydrophillic - mask these proerties by pairing with H bonds

Question 18

what is a globular protein that is an exception to the hydrophobic effect?

Answer 18

porins - hydrophobic residues on the outside to interact with alkane chains
charged/ polar AA surround water-filled channels

Question 19

side-chain interactions - covalent?

Answer 19

covalent interactions are disulfide bonds
- rarely found in the cell due to high glutathione and beta mecaptoethanol (good reducing agents)

Question 20

side chain interactions - non-covalent?

Answer 20

ionic, hydrogen, hydrophobic interaction

Question 21

side chain interactions.

Answer 21

VDW - random electron movement forms dipoles

Question 22

how is active ribonuclease converted to its inactive form?

Answer 22

add beta mecaptoethanol (reducing agent- reduce disulfide bonds)
add 8M urea (chaotropic agent) - breaks up hydrogen bonding and non cov interactions

Question 23

the

what happens if both BM and 8M urea are added to inactive ribonuclease?
(reason why on separate flashcard)

Answer 23

the polypeptide seq of the denatured ribonuclease will only fold back into the active formation (and not another conformation)

Question 24

what happens if only 8M urea is added to inactive ribonuclease?

Answer 24

forms many scrambled ribonuclease structures (not the active conformation) -

this is because urea stops interactions between side chains so the correct cysteine’s are not placed in the correct proximity
therefore, final protein structure cannot form

Question 25

why does the active conformation form when both BM and 8M urea are added?

Answer 25

BM reduce the disulfide bonds of the incorrect cysteine's (as incorrect one is not strong enough and is reduced) this continues until the correct cysteine's match there is also a decrease in gibbs free energy as the scrambled ribonucleases are converted to the stable native conformation - more energetically favourable

Question 26

co-operative transition of ribonuclease formation (and many other proteins)

Answer 26

only 2 conformations present to any significatn extent (denatured and stable) protein folding is an all-or-none process

Question 27

fibrous protein - alpha keratin

Answer 27

coiled-coil proteins (2 or more alpha helices entwine, held by VDW) alpha helices can stretch - break VDW covalent covalent disulfide bond - return the fibre to its original shape

Question 28

fibrous protein - collagen

Answer 28

3 helical ppd chains glycine - every 3rd residue (small, so fits in interior) proline/hydroxyproline - cause steric repulsion that stabilises the helix H bonds betwen adjacent chains holds the ppd chains together

Question 29

quaternary structure: - simplest? - more than 2 identical subunits?

Answer 29

homodimer (2 identical subunits) more than 2 identical subunits = oligomeric - the subunits in an oligomeric protein are called protomers

Question 30

what is the proteome

Answer 30

entire complement of proteins expressed in a cell we can increase proteome with post-translational modifications

Question 31

2 proteins on the surface of the flu particle?

Answer 31

Neuraminidase (N) (tetrameric structure, 4 subunits) haemagglutinin (H)

Question 32

how do haemaglutinin and neuraminidase work to allow virus to infect many cells?

Answer 32

1. H has high affnity to sialic acid (a sugar monomer part of a glycosylated protein) - present on epithelial cells in nose = interaction between epithelial cell and viral particle 2. interaction holds virus in close proximity -enters cell (endocytosis) 3. viral replication and budding - fuse back with cell to be released 4. once released, H recognises sialic acid on the cell surface again, trapping virus 5. neuraminidase - cleaves the sialic acid = vrius escapes

Question 33

what drug would stop virus spreading (in terms of H and N)

Answer 33

drug that is an inhibitor of neuriminidase stops sialic acid binding to active site and being cleaved virus remains trapped

Question 34

what is a conjugated protein?

Answer 34

contains permanently associated chemical components as well as their AA non AA part = prostethic group

Question 35

steps of protein purification?

Answer 35

1. transformation -ligated plasmid uptaken 2. selection = AB exposure. Ones that survive contain the ligated plasmid 3. cell growth and protein production 4. cell lysis 5. chromatography to extract protein 6. dialysis - separate from smaller solutes 7. SDS-PAGE - assess purity 8. protein assay - test for protein function

Question 36

how does an SDS-PAGE assess for purity?

Answer 36

pure - single, well-defined band at the expected molecular weigth impure - multiple bands that indicate the presence of contaminants

Question 37

what is gel filtration? what is another name for it?

Answer 37

separate on molecular size - column of porous beads - small proteins can enter the beads so travel slower through the column Another name is size exclusion chromatography

Question 38

chromatography types -ion exchange

Answer 38

separate molecules based on molecular charge can have +ve and -ve resin (charged molecules will bind to resin if its oppositely charged)

Question 38

chromatorgraphy types - affinity chromatography

Answer 38

uses specific binding to separate molecules immbolised ligand onto a solid matrix only 1 protein in the mixture has affinity to the ligand wash buffer - removes contaminant proteins that are not bound to ligand elution buffer -remove desired protein by breaking specific interactiosn

Question 39

which chromatography techique is a His-tag used for and how does it work?

Answer 39

used in affinity chromatography to separate a protein with an unknown structure attach His-tag to the desired protein stationary phase = immobilised nickel resin proteins with His-tag will interact with and bind to the nickel elution buffer is imidazole, which disrupts the interaction between the His-tag and nickel resin, causing elution of the protein

Question 40

what is a protein assay and give an example?

Answer 40

check for protein activity for example, check the activity of the enzyme lacate dehydrogenase lactate <=> pyruvate converts NAD+ -> NADH NADH absorbs light at 340nm measure the increase in light absorption at 340nm over time