7b

Question 1

what is fibroin and what type of protein is it

Answer 1

its the thing that makes spider silk

its a fibrous protein : insoluble protein
antiparallel beta pleated sheets

Question 2

fibroin,, the spider silk fibrous protein has a high abundance of glycine,, what does a high number of glycine in a ppc do

Answer 2

it allows for tight packing of sheets by allowing the r groups to interlock allowing silk to be strong and flexible

Question 3

what are proteases and what type of protein are they

Answer 3

proteases are globular proteins and theyre enzymes that cleave peptide bonds

Question 4

what favourability does peptide hydrolysis have

Answer 4

it is thermodynamically favoured but not kinetically favoured bc it takes like 300 years for it to occur

Question 5

what do proteases do

Answer 5

they digest food and help ur blood clot

Question 6

what is kenesis

Answer 6

its a motor protein in eukaryotic cells

its movement supports cellular functions like cell division

Question 7

brief explanation on determining the primary structure

Answer 7

• split the ppc ( break disulphide, hydrogen, electrostatic etc)
• determine the aa composition + the relative amount of each one in each subunit
• cleave the subunits into smaller oligopeptides (10 aa)
• determine the sequence of aa in protein

Question 8

how can we break a disulphide bond? in order to split the ppc

Answer 8

we go from disulphide to 2 thiols using mercaptoethanol

this is a reduction reaction
( ethane with alcohol on one end and thiol on the other )

we then react the thiols with a carboxylate with an methyl iodine on one end.

the thiols act as a nucleophile and attack the electrophilic CX bond to kick off the I.

this prevents the thiols from forming a disulphide bond again

Question 9

how can we hydrolyse amide bonds

Answer 9

we have the ppc and we put the ppc in 6M HCl ,, 100-110*C for 24 hours

Question 10

whats bad about hydrolysing a ppc in 6M HCl,, 100-110*C for 24 hours

Answer 10

some aa get degraded!!! aka partially destroyed

Question 11

what do we do once we hydrolyse the amide bonds using 6M HCl,, 24 hours at 100-110

Answer 11

we get separated aa which we can then analyse by ion exchange chromatography or reversed phase high pressure liquid chromatography.

Question 12

whats ion exchange chromatography

Answer 12

when charged aa are attracted to charged resin.

Question 13

how can we determine the quantity of aa

Answer 13

we use uvvis spec + form a metal complex to use the uvvis spec on

or we can use ninhydrin bc it turns pink ,, but we only use ninhudrin if theres more than 10nmol of aa present

Question 14

whats bradykinin and how can we tell what aa make it up

Answer 14

bradykinin is an inflammatory mediator

• heat + HCl to hydrolyse amide bonds
• put on chromatography machine

u have the a reference graph of abs on the y axis and time on the x axis. the reference has the peaks for each aa

u then analyse the aa u have by looking at the time of abs and the abs and u compare this with the reference to identify the aa in ur sample!!!

this doesnt tell us about the sequency tho,, it tells us what amino acids are present

Question 15

what chemical reagents do we use to do N terminus determination

Answer 15

• SANGER ( sankar)
• DANSYL ( Danny)
• EDMAN ( redman)

Question 16

whats the main thing we do with N terminus determination with all of the chem reagents

Answer 16

• sanger
• dansyl
• edman

bc the n terminus is usually NH3+.,, we find a pH that passes its pka to form NH2

bc NH2 is neutral meaning it has its lone pair meaning its nucleophilic and can attack electrophiles and form bonds to them.

we use the chemical reagents as electrophiles to use them as ‘labels’ that attach to that n terminus aa!!!

Question 17

describe sangar chemical reagent

Answer 17

1, fluoro, 2,4 - dinitrobenzene

the CF gets attacked by the NH2

Question 18

describe dansyl chloride

Answer 18

2 benzenes fused together

SO2Cl on the bottom of the 1st benzene

N - dimethyl at the top of the 2nd benzene

pretty sure the C is electrophilic in ncs

the Cl is the leaving group

Question 19

describe edman chemical reagent

Answer 19

benzene with N=C=S on the top of the benzene

the C is electrophilic

Question 20

sanger not only binds to the n terminus but also what

Answer 20

any nh2 in the side chains

Question 21

describe the spranger reaction mechanism

Answer 21

• the end amine attacks the C of the CF bond and kicks off F
• hydrolysis then occurs using H3O+ to break the rhs amide bond and give u a carboxylic acid.

Question 22

what do the nitro groups on the spranger do

Answer 22

they activate the benzene and allow nucleophilic aromatic substitution to occur!!!

Question 23

describe the dansyl reaction mechanism

Answer 23

• its the best choice to use when the amount of protein u have is limited
• makes the n terminus amino acid glow // flourescence.
• the amine attacks the S of the CO2Cl and kicks of the Cl

u then use HCl and a high temo to hydrolyse the peptide bonds and form a carboxylate ion on the rhs when u break the n terminus aa from the peptide

Question 24

how do we know the exact aa sequence in a protein

Answer 24

• the N and C terminus are established by a sequence of reactions specific for those aa
• the large proteins are turned into smaller fragments using site specific chemical reagents // endopeptidases
• the segments are then separated and sequenced
• the procedure is repeated using diff enzymes that cleave at different sites / cleave different amino acids
• we compare the first and second fragment groups and overlap them to see how they acc form the overall ppc.

Question 25

CNBr,, what bond does it cleave

Answer 25

it cleaves the carboxyl side of the methionine residues

Question 26

Hydroxylamine,, what bond does it cleave

Answer 26

asparagine - glycine bonds

Question 27

o-iodosobenzoate,, what bonds does it cleave

Answer 27

carboxyl side of tryptophan residues. tryptophan is not a common aa

Question 28

2-nitro-5-thiocyanobenzoate,, what bonds does it cleave

Answer 28

it cleaves the amino side of cysteine residues

Question 29

describe CNBr cleavage mechanism

Answer 29

it cleaves methionine only!! methionine has a SCH3 side chain!! the S acts as a nucleophile an uses its lone pair to attack the C of CNBr which then kicks off the Br. the N on the RHS of the moolecule then uses its lone pair to form a double bond and resonate onto the carbonyl its bonded to. the ocygen of this carbonyl then attacks the 2nd BH2,, aka the CH2 closest to the S the e- are used to neutralise the S(+) and kick off that part of the moelcule!! u then have like a little triangle moment between the O of the carbonyl and the CH2 u attacked u then undergo imine hydrolysis using water to form a carbonyl and kick of the amine partttttt

Question 30

describe enzymatic hydroldescribe the 2 enzymes and what bonds they cleave in enzymatic hydrolysis

Answer 30

u have - trypsin: cleaves at the c terminus side of basic aa lys + arg : gives ammonium and carboxylate ions - chymotrypsin : cleaves at the c terminus side of aromatic aa like phe, tyr and Trp : these are greasy aa.

Question 31

describe edman degradation

Answer 31

- cuts off the n terminus aa one by one!!! - u cut of the aa and obvs the reagent is stuck to it so the aa is always labelled - its then analysed - so u get sequential identification of aa from the n terminus to c terminus side of ppc

Question 32

what reagent is used in edman degradation

Answer 32

PTC phenyl isothiocyanate reagent Ph - N = C = S NCS but with a ph attached to it.

Question 33

how many aa can u use the edman degradation on

Answer 33

up to 50!! it gets less accurate after that

Question 34

whats good about edman degradation

Answer 34

that once u remove the aa,, the rest of the peptide chain is still intact

Question 35

describe the edman degradation mechanismmmmm

Answer 35

the N teminus NH2 attacks the C of the phNCS and the e- move onto the S the s then uses its lone pair to reform the C=S bond and the N=C bond attacks one of the H's on the NH2 to form a single bond the ph NCS kinda turns a bit so its parallel kinda to the aa. the S can then attack the C carbonyl on the amide bond the carbonyl is protonated btw!! and the couble bond of the crabonyl neutralises the O+ on the carbonyl while the N on the NCS which has a H uses its lone pair to reform the CN double bond on the ph NCS molecule. this then forms a ring a h20 then removes the H on the OH and reforms the carbonyl we previously attacked. and the amine part of the aa is kicked off. u then add HCl ( for the back reaction u use heat) and u get a cute molecule with C=S at the top,, NH on the LHS and NPh on the RHS a CRH on the LHS and a carbonyl on the RHS we then analyse to find what the R group is using chromatography by comapring the retention time of the compound being analysed and a set of standard compounds when R is the side chain of the aa.

Question 36

ehats the overlap peptide strategy

Answer 36

u have a peptide and CNBr ,, u then get 2 peptides of made up of different aa. u then take the same type of peptide and use a diff enzyme aka trypsin,, ajs this also forms 2 peptides but with combos of different aa. u then compare the amino acids in each 4 peptides and join them up in a way that makes most sense!! aka where they overlap to get the overal peptide sequence.