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O+B > Amino acids, Peptides and Proteins > Flashcards

Amino acids, Peptides and Proteins Flashcards

1
Q

only R amino acid

A

L-cysteine
R= CH2CH2SH
structural implications: forms covalent bonds with itself

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2
Q

only achiral amino acid

A

Glycine
minimises steric effects in proteins
R=H2

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3
Q

Stecker synthesis reagents

A
  1. NH3, HCN
    NH3 and CN- both attack carbonyl carbon
  2. H+/H2O
    H2O breaks C-N triple bond and attacks carbonyl carbon
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4
Q

Enantioselective hydrolysis

A

add acyl chloride and NaOH. Cl replaced with Nitrogen
Porcine kidney acylase - chiral enzyme so only hydrolysis’ one enantiomer
Separation by differential solubility or ion exchange chromatography

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5
Q

peptide bond: phi angle (O with line through)

A

angle between the carbonyl groups through bond N-alpha-C to CO (OC-NH-CRH-CO)
bond around which there is free rotation i.e. single bonds

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6
Q

peptide bond: psi angle (schrodinger symbol)

A

angle between amine groups through CO and alpha-C to nitrogen (HN-CRH-CO)
bond around which there is free rotation i.e. single bonds

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7
Q

The Dihedral Angles of Rotation for an Amino acid in a Peptide chain: right handed (RH) alpha-helix

A 
phi = -57
psi = -47
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8
Q

The Dihedral Angles of Rotation for an Amino acid in a Peptide chain: parallel beta-sheet

A 
phi = -119
psi = 113
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9
Q

The Dihedral Angles of Rotation for an Amino acid in a Peptide chain: antiparallel beta-sheet

A 
phi = -139
psi = 135
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10
Q

Non-polar amino acids

A

R group non-polar
9 non-polar amino acids
Glycine R=H2
L-Alanine R=CH3

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11
Q

Forces involved in protein-protein as well was substrate-protein interactions : Hydrophobic interactions

A

Increasing chain length and branching increases added hydrophobic bonding capacity
2 examples = L-alanine R=CH3 +2.85 kJ mol-1 (lowest)
L-Phenylalanine R= CH2Ph +15 kJ mol-1 (highest)
non-polar amino acids
hydrophobic core of a protein

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12
Q

Polar neutral amino acids

A

L-cysteine R = CH2SH (R- configuration)

L-serine R=CH2OH

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13
Q

Polar charged amino acids: basic

A

L-Lysine R = CH2CH2CH2CH2NH2 (C4H9NH2)

pKa = 9

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14
Q

Polar charged amino acids : acidic

A

L-Glutamic acid R= CH2CH2COOH pKa=4
L-Aspartic Acid R = CH2COOL pKa = 4
differ by one -CH2

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15
Q

Forces involved in protein-protein as well was substrate-protein interactions : Hydrogen Bonding

A

donor and acceptor
donors = hydrogens attached to EN elements
acceptors = EN elements with l.p.

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16
Q

Forces involved in protein-protein as well was substrate-protein interactions - list them

A
  1. Hydrogen bonding - 7.5 kJ mol-1 distance 2A
  2. Hydrophobic Interaction - up to 15 kJ mol-1
  3. Salt Bridges - 25-50 kJ mol-1 distance 2-3A
  4. Cation/Pi interactions - 4-10 kJ mol-1 distance 4-10 A
  5. Van der Waals forces - 6-8 kJ mol-1 2-4 A
  6. Covalent interactions
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17
Q

Forces involved in protein-protein as well was substrate-protein interactions : Salt Bridges

A

anions and cations
anions - O- (negatively charged oxygen/ other EN elements (?))
cations - N+ (positively charged nitrogen or metals e.g. Mn2+, Mg2+, Li+)

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18
Q

Forces involved in protein-protein as well was substrate-protein interactions : Covalent protein-protein interactions

A

Cysteine is the only amino acid with side chains (R=CH2SH) that can covalently bond
disulfide bond formed through oxidation

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19
Q

Forces involved in protein-protein as well was substrate-protein interactions : cation/pi interactions

A

parallel stacking interactions
e.g. phenyl groups stacked, delocalised positive charge parallel to phenyl group
perpendicular interaction
e.g. delocalised positive charge perpendicular to penyl group

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20
Q

primary protein structure

A

defined simply as the linear sequence of amino acids
all the information required to figure out the structure on the protein as well as define the catalytic activity of the enzyme is contained within the primary sequence
R groups alternate charges away from each other
must know primary sequence in order to: 1. determine the protein structure 2. determine the mechanism of action of the enzyme

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21
Q

secondary protein structure

A

defined as the local spatial arrangement of the main chain atoms
the primary structure spontaneously folds into local regions of structure which may comprise 6-20 amino acids

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22
Q

3 main secondary structural elements

A

alpha-helix
beta-sheet
random coil

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23
Q

alpha-helix

A

formed from a single peptide
resembles a coiled spring
right-hand (clockwise) turn
stabilised by H-bonding between carbonyl oxygen of one amino acid and the amide hydrogen of the residue 4 amino acids ahead in the primary sequence
3.6 amino acids in each turn of the helix
pitch is 5.4 A
R groups point out and slightly backwards from the helix, side chains 4 ahead will be close in space

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24
Q

why does a alpha-helix form

A

driven by primary sequence
when the amino acids are coiled up, all the hydrophobic residues line up on one side and all of the polar residues on the other

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25
Q

beta-sheet

A

beta-pleated: rippled or pleated effect of the polypeptide chain from a side view
partial double bond character of the amide bond
side-chain R groups are trans
successive side-chains extend from opposite side of the beta-sheet
sometimes: one side side-chains hydrophobic, the other polar

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26
Q

beta-sheet: antiparallel or parallel

A 
anti: arrows alternate up and down 
H bonding straight 
parallel: arrows one way 
H bonding at an angle 
H bonding holds structure together
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27
Q

random coil

A

non-structured polypeptide regions which link either alpha-helices or beta-sheets (although could be something else)
no stabilised structure

28
Q

tertiary protein structure

A

the arrangement in space of all atoms in a single polypeptide - the 3D arrangement of the secondary structures
folding driven by burying and clustering of hydrophobic side chains to minimise water contact
small stretches of secondary structure act as nuclei for the stabilisation of other structures
protein grows in cooperative fashion
folding units form, condense, and the form larger folding units

29
Q

quaternary protein structure

A

some proteins subunits must associate in geometrically specific patterns in order to confer catalytic activity on the complex
advantages (of this)
1. defects in subunits can be repaired by replacing individual subunits
2. the active protein can be assembled at a site which is different from where it is manufactured
3. sub-units can be self assembling, thus there is less genetic information required
4. can have more than one active site on a protein
5. subunit construction allows regulation

30
Q

the proximity (propinquity) effect

A 
intramolecular catalysis - reactions in an enzyme substrate complex are first order 
catalytic groups acting cooperatively on the same molecule
catalytic groups (unimolecular) have higher effective concentration than bimolecular reactions in solution 
intramolecular Nu- held more rigidly with respect to reaction centre (hydrophobic environment) , rotational and translational entropies lost on binding and not during subsequent catalytic steps
intramolecular nu- much less heavily solvated than intermolecular nu- in dilute solution
31
Q

nucleophilic catalysis examples

A

L-cysteine R=CH2SH

L-serine R= CH2OH

32
Q

nucleophilic catalysis

A

potent as desolated in active site
Nu- form covalent bonds between enzyme and substrate to give a reactive intermediate
reaction intramolecular
bonding must be reversible so products can leave the active site

33
Q

electrophilic catalysis

A

a covalent intermediate is formed between the cationic E+ associated with the enzyme and an electron rich portion of a substrate molecule
no very effective E+ amino acid side chains - not important
most commonly electron deficient organic cofactors e.g. vitamins B6 and B1

34
Q

polar acidic amino acids

A

deprotonated above pKa

35
Q

polar basic amino acids

A

protonated below pKa

36
Q

general acid

A

AH stabilises -ve charge of RCOOR

37
Q

general base

A

B stabilises +ve charge of RCOOR…H2O+

38
Q

concerted acid-base

A

+ve charge stabilised by base, -ve by acid

enzymes

39
Q

acetylcholineesterase

A

bimolecular rate coefficient ~ rate of diffusion
breaks down amino acid (CH3COOCH2CH2NMe3+) into acetate (CH3COO-) and choline (HOCH2CH2NMe3+)
enz-base attacks H2O which attacks C=O
bonds to esteratic site, broken down and then released

40
Q

acetylcholinesterase mechanistic steps

A
  1. enzyme-substrate complex
  2. tetrahedral intermediate
  3. acyl enzyme intermediate - alcohol of choline formed
  4. alcohol leaving group replaced by water from solvent - general base goes through water to attack acyl enzyme intermediate
  5. tetrahedral intermediate
  6. active enzyme - product diffuses out of active site
41
Q

what stops acetylcholinesterase

A

Sarin and VX

bind to serine and cant hydrolyse strong O-P bond

42
Q

importance of TS stabilisation

A

to achieve optimal catalysis enzymes should selectively bind the TS rather than the substrate
no advantage for enzyme to bind tightly
binding constants for enzymes - milli to micro molar range
binding constants for binding proteins and antibodies whose function is to bind small molecules tightly - nano to picomolar range
stabilising TS reduces activation energy so enzyme works faster

43
Q

the lysozyme mechanism: Koshland

A

SN2
covalent intermediate
preferred mechanism

44
Q

the lysozyme mechanism: Phillips

A

carbocations

probably wrong

45
Q

DCC

A

cyclohexane-N=C=N-cyclohexane
used to couple an amine and acid group to form an amide efficiently and under mild conditions
dicyclohexylurea - very insoluble (disadvantage)

46
Q

DIC

A 
diisopropylcarbodiimide 
DIC 
(CH3)2CH-N=C=N-CH(CH3)2
soluble urea 
activates acid to form amino acid
unwanted side reaction: O-acylisourea rearranges to N-acylurea before desired attack
47
Q

2nd generation coupling reagent

A

DIC + HOBt (1-hydroxybenzotriazole)
HOBt more nucleophilic than amine
HOBt forms a 2nd activated ester intermediate
HOBt - catalyst

48
Q

DIPEA/BOP strategy

A 
DIPEA deprotonates COOH 
BOP forms activated amino acid 
BOP contains HOBt 
loss of HMPA 
BOP robust
49
Q

Protecting groups

A

used against self condensations in polycondensations
COOH: Wang linker (CH2PhO-solid support) attached to solid support - acid labile attachment of COOH to solid support
NH: Fmoc, base labile amine protecting group - 20% piperidine in DMF (end of amino acid) used at each extension amino acid
t-Boc - protection of amine side chains. acid labile 25-50% TFA in DCM
t-Bu - protection of alcohols and carboxylic acids - 90% TFA in DCM

50
Q

Wang Linker

A

CH2PhO-solid support
starting C terminal amino acid - attachment of carboxylic acid to solid support
acid labile

51
Q

Fmoc

A 
amine protecting groups 
used for each extension of amino acid 
base labile 
20% piperidine in DMF (solid phase) 
deprotection - form carbamic acid and then lose CO2
52
Q

t-Boc/Boc

A 
amine protecting group side chains 
acid labile 
25-50% TFA in DMF 
deprotection - scavenger ion needed 
TFA/H+ turns amide to NH2 and CO2
53
Q

t-Bu / Bu

A

protection of alcohols and carboxylic side chains
acid labile
90% TFA in DMF
hardest to remove - strong acid
add scavenger to stop carbocation undergoing unwanted side reactions with certain amino acid side chains

54
Q

solid phase peptide synthesis

A

precipitation from diethyl ether
analysis by MS and HPLC
Purification by HPLC

55
Q

acid sensitive linkers

A

operate via formation of a highly resonance-stabilised benzyl cation. protonation of the acid/amide carbonyl followed by movement of the benzyl electron pair to the carbonyl/amide results in cleavage of the product from the resin. leaving linker as a cation
the more resonance forms and electron donating substitutients available to the benzyl cation the more acid-sensitive the linker

56
Q

which aromatic amino acids absorb significantly at 280 nm? (UV)

A

tryptophan (W) and tyrosine (Y)

appreciable extinction coefficients at 280 nm

57
Q

UV - estimate protein conc using A280

A

A280 arises from aromatic amin acid side-chains
A280 = e280.c.l
c = conc l=length (usually 1)
three scenarios
1. e280 is known - conc can be calculated
2. e280 not known but can be calculated by measuring A280 on a known amount of protein or using a calibration curve
3. approximate using e280 = (no. Trp)(5500) + (no. tyr)(1490) + (no cys)(125)

58
Q

Circular Dichroism (CD)

A

used to detect chiral molecules and structures.
rapid alternation of right and left-circularly polarised light
chiral molecules absorb at different degrees

59
Q

Circularly polarised light

A

two polarised waves travelling at right angle to each other, appears to rotate when observed

60
Q

CD spectrum

A

delta E curve ( lowest energy) given in different signs with enantiomers
elipicity vs wavelength
alpha - minima - 209 and 222 nm maxima 192 nm
beta sheet- minma 218 nm maxima 196 nm
coil - minima 195-6 nm maxima 212 nm (opposite to other two )

61
Q

soft ionisation

A

electron spray ionisation (ESI)
low energy
no fragmentation of the analyte

62
Q

hard ionisation

A

chemical ionisation (CI)
high energy
causes fragmentation of analyte

63
Q

ESI

A

capillary tip - +ve charge
analyte molecule becomes multiply +ve charged droplet
solvent evaporates - molecule is smaller (Rayleigh limit)
coloumbic explosion - charged, gas phase analyte molecules (smaller, droplet broken up)

64
Q

What could go wrong with ESI (difference between expected and observed)

A

imperfect deprotection
incorrect sequence
disulfide bond formation

65
Q

calculating protein MW from adjacent charge states

A 
X= (M + Zx)/Zx
Y=(M + Zx +1)/Zx + 1 
Zy = Zx +1 
Zx = (Y-1)/(X-Y) 
M = (X*Zx)-Zx = (Y*Zy) - Zy
66
Q

deconvolution

A

removes unnecessary/complicated peaks?

O+B (5 decks)

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