Amino acids, Peptides and Proteins Flashcards
only R amino acid
L-cysteine
R= CH2CH2SH
structural implications: forms covalent bonds with itself
only achiral amino acid
Glycine
minimises steric effects in proteins
R=H2
Stecker synthesis reagents
- NH3, HCN
NH3 and CN- both attack carbonyl carbon - H+/H2O
H2O breaks C-N triple bond and attacks carbonyl carbon
Enantioselective hydrolysis
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
peptide bond: phi angle (O with line through)
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
peptide bond: psi angle (schrodinger symbol)
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
The Dihedral Angles of Rotation for an Amino acid in a Peptide chain: right handed (RH) alpha-helix
phi = -57 psi = -47
The Dihedral Angles of Rotation for an Amino acid in a Peptide chain: parallel beta-sheet
phi = -119 psi = 113
The Dihedral Angles of Rotation for an Amino acid in a Peptide chain: antiparallel beta-sheet
phi = -139 psi = 135
Non-polar amino acids
R group non-polar
9 non-polar amino acids
Glycine R=H2
L-Alanine R=CH3
Forces involved in protein-protein as well was substrate-protein interactions : Hydrophobic interactions
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
Polar neutral amino acids
L-cysteine R = CH2SH (R- configuration)
L-serine R=CH2OH
Polar charged amino acids: basic
L-Lysine R = CH2CH2CH2CH2NH2 (C4H9NH2)
pKa = 9
Polar charged amino acids : acidic
L-Glutamic acid R= CH2CH2COOH pKa=4
L-Aspartic Acid R = CH2COOL pKa = 4
differ by one -CH2
Forces involved in protein-protein as well was substrate-protein interactions : Hydrogen Bonding
donor and acceptor
donors = hydrogens attached to EN elements
acceptors = EN elements with l.p.
Forces involved in protein-protein as well was substrate-protein interactions - list them
- Hydrogen bonding - 7.5 kJ mol-1 distance 2A
- Hydrophobic Interaction - up to 15 kJ mol-1
- Salt Bridges - 25-50 kJ mol-1 distance 2-3A
- Cation/Pi interactions - 4-10 kJ mol-1 distance 4-10 A
- Van der Waals forces - 6-8 kJ mol-1 2-4 A
- Covalent interactions
Forces involved in protein-protein as well was substrate-protein interactions : Salt Bridges
anions and cations
anions - O- (negatively charged oxygen/ other EN elements (?))
cations - N+ (positively charged nitrogen or metals e.g. Mn2+, Mg2+, Li+)
Forces involved in protein-protein as well was substrate-protein interactions : Covalent protein-protein interactions
Cysteine is the only amino acid with side chains (R=CH2SH) that can covalently bond
disulfide bond formed through oxidation
Forces involved in protein-protein as well was substrate-protein interactions : cation/pi interactions
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
primary protein structure
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
secondary protein structure
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
3 main secondary structural elements
alpha-helix
beta-sheet
random coil
alpha-helix
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
why does a alpha-helix form
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
beta-sheet
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
beta-sheet: antiparallel or parallel
anti: arrows alternate up and down H bonding straight parallel: arrows one way H bonding at an angle H bonding holds structure together
random coil
non-structured polypeptide regions which link either alpha-helices or beta-sheets (although could be something else)
no stabilised structure
tertiary protein structure
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
quaternary protein structure
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
the proximity (propinquity) effect
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
nucleophilic catalysis examples
L-cysteine R=CH2SH
L-serine R= CH2OH
nucleophilic catalysis
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
electrophilic catalysis
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
polar acidic amino acids
deprotonated above pKa
polar basic amino acids
protonated below pKa
general acid
AH stabilises -ve charge of RCOOR
general base
B stabilises +ve charge of RCOOR…H2O+
concerted acid-base
+ve charge stabilised by base, -ve by acid
enzymes
acetylcholineesterase
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
acetylcholinesterase mechanistic steps
- enzyme-substrate complex
- tetrahedral intermediate
- acyl enzyme intermediate - alcohol of choline formed
- alcohol leaving group replaced by water from solvent - general base goes through water to attack acyl enzyme intermediate
- tetrahedral intermediate
- active enzyme - product diffuses out of active site
what stops acetylcholinesterase
Sarin and VX
bind to serine and cant hydrolyse strong O-P bond
importance of TS stabilisation
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
the lysozyme mechanism: Koshland
SN2
covalent intermediate
preferred mechanism
the lysozyme mechanism: Phillips
carbocations
probably wrong
DCC
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)
DIC
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
2nd generation coupling reagent
DIC + HOBt (1-hydroxybenzotriazole)
HOBt more nucleophilic than amine
HOBt forms a 2nd activated ester intermediate
HOBt - catalyst
DIPEA/BOP strategy
DIPEA deprotonates COOH BOP forms activated amino acid BOP contains HOBt loss of HMPA BOP robust
Protecting groups
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
Wang Linker
CH2PhO-solid support
starting C terminal amino acid - attachment of carboxylic acid to solid support
acid labile
Fmoc
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
t-Boc/Boc
amine protecting group side chains acid labile 25-50% TFA in DMF deprotection - scavenger ion needed TFA/H+ turns amide to NH2 and CO2
t-Bu / Bu
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
solid phase peptide synthesis
precipitation from diethyl ether
analysis by MS and HPLC
Purification by HPLC
acid sensitive linkers
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
which aromatic amino acids absorb significantly at 280 nm? (UV)
tryptophan (W) and tyrosine (Y)
appreciable extinction coefficients at 280 nm
UV - estimate protein conc using A280
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)
Circular Dichroism (CD)
used to detect chiral molecules and structures.
rapid alternation of right and left-circularly polarised light
chiral molecules absorb at different degrees
Circularly polarised light
two polarised waves travelling at right angle to each other, appears to rotate when observed
CD spectrum
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 )
soft ionisation
electron spray ionisation (ESI)
low energy
no fragmentation of the analyte
hard ionisation
chemical ionisation (CI)
high energy
causes fragmentation of analyte
ESI
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)
What could go wrong with ESI (difference between expected and observed)
imperfect deprotection
incorrect sequence
disulfide bond formation
calculating protein MW from adjacent charge states
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
deconvolution
removes unnecessary/complicated peaks?
