PROTEINS

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PROTEINS ENZYMES
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PROTEINS
BIOLOGICAL ROLES Structure -
collagen (skin), keratin (hair) Transport
- membrane transporters Enzymes
- catalysts Motility -
myosin Immune system - Ig Metabolic
regulation - hormones
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DNA mRNA PROTEIN
DNA
mRNA
Peptide/protein
AA AA AA AA..
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1. Structure of AAs - L amino acids
2. Different types of amino acids - R group
3. Amino acid modifications
4. Ionisation of AAs
5. Peptide bond
6. Biologically important peptides

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AMINO ACID STRUCTURE
?-carbon
H H2N C COOH
R
amino group
carboxyl group
Side chain
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L-amino acids make up proteins
mirror plane
H H2N C COOH
R
H HOOC C
R
NH2
L- amino acid
D-amino acid
Enantiomers
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Side chain (R ) determines AA properties
HYDROPHOBIC (and aliphatic) R (non-polar)
H
CH2 CH CH3 CH3
CH CH3 CH3
CH3
Glycine (G)
Alanine (A)
Valine (V)
Leucine (L)
Also Isoleucine (I), methionine (M), proline (P)
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HYDROPHOBIC (and aromatic) R (non-polar)
CH2 C CH NH
CH2
Phenylalanine (F)
Tryptophan (W)
Also Tyrosin (Y)
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HYDROPHILIC (and charged) R (polar)
CH2 CH2 CH2 CH2 NH3
CH2 CH2 C O O
CH2 C O O
Aspartate (D)

Glutamate (E)
Lysine (K)
Also Arginine (R ) and histidine (H) ve charge
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HYDROPHILIC (and neutral) R (polar)
CH2 CH2 C O NH2
CH2 OH
CH2 C O NH2
Serine (S)
Asparagine (N)
Glutamine (Q)
Also Threonine (T) and Cysteine (C)
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Amino Acid properties
HYDROPHILIC R groups - H bonding with water In
aqueous environment on OUTSIDE of
protein HYDROPHOBIC R groups -Hydrophobic
interactions In aqueous environment on INSIDE of
protein
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PROTEIN IN AQUEOUS ENVIRONMENT
hydrophilic
hydrophobic
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Special Features of some Amino acids
Methionine - 1st amino acid in protein sequence
(N-terminal) Cysteine (SH grp) - disulphide
bond formation Aromatic amino acids
(tryptophan, tyrosine, phenylalanine)
UV absorption at 280nm
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AMINO ACID MODIFICATION
Serine and threonine (OH grp) -O-linked
glycosylation (glycoprotein) -
reversible phosphorylation (ser/thr
kinases) Asparagine (NH2 grp) - N-linked
glycosylation Proline (ring) - forces bend in
protein structure - can
be hydroxylated (e.g. in collagen)
20 amino acids coded, many more from modification
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IONISATION STATE OF AMINO ACIDS
H H2N C COO H
H H3N C COO H
H H3N C COOH H


pH 1 charge
pH 7 no net charge
pH 11 - charge
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H H3N C COO H
ISOELECTRIC POINT pI
pH when no net charge

pI7
Aspartate
H H3N C COO CH2
COO
H H3N C COO CH2
COOH


pH 7 net -ve charge
pH 4 no net charge (pI)
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R group ionisation
ACIDIC Glutamate, aspartate - R grp COO- at
neutral pH pI lt 7 BASIC Lysine, arginine -
R group NH3 at neutral pH pI gt 7
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PEPTIDE BOND FORMATION
R1 NH2 CH COOH
R2 NH2 CH COOH

R1 H2N CH C
R2 NH CH COOH
O
H2O
Peptide bond
Dipeptide
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PEPTIDE UNIT IS RIGID
Peptide unit
R1 H2N CH C
R2 N CH COOH

O
H
Partial double bond character
Rotational freedom
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PROTEIN 3D STRUCTURE
RIGID PEPTIDE BOND - well
defined structure ROTATIONAL FREEDOM
- proteins can fold in many ways
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POLYPEPTIDE CHAIN
Dipeptide
tripeptide
polypeptide
R1 NH2 CH C
R2 NH CH C
R3 NH CH C
R4 NH CH COOH
O
O
O
Amino terminal residue
Carboxyl terminal residue
Protein sequence from amino to carboxyl end
N C
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NATURAL PROTEINS
Between 50 - 2 000 amino acid residues Mean
molecular mass of amino acid residue 100
Daltons Mean molecular mass of protein 5 000
to 200 000 Daltons
5 to 200 kiloDaltons
(kDa)
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3D STRUCTURE OF PROTEINS
PEPTIDES POLYPEPTIDES PROTEINS
Increasing complexity of structure
4 different levels of protein structure
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1y structure
AMINO ACID SEQUENCE
Only interaction btw AAs is peptide bond No other
interactions or stabilising bonds
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2Y STRUCTURE
Local structure of polypeptide chain H BONDING
between carbonyl oxygen and amide hydrogen
R1 NH2 CH C
R2 N CH C
R3 N CH C
R4 N CH C
O
O
O
O
H
H
H
O
O
O
R5
R6
R7
C CH N C CH N C CH N
H
H
H
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2 TYPES OF 2y STRUCTURE

• Alpha helix (?-helix)
• Rod-like structure
• Coiled peptide chain
• R groups extend outwards from axis
• H bonding btwn CO and NH 4 residues away
• 3.6 amino acids per turn of helix

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Model of ?-helix
H bonds CO HN
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?- helix in proteins
myoglobin and haemoglobin 75 alpha-helix
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? -helical coiled coils
2 or more ?-helices entwined - very strong eg
Myosin, tropomyosin in muscle Fibrin in
blood clots Keratin in hair
SUPERHELIX
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2. Beta sheets (?-sheets) Polypeptide chain
is extended H bonding between CO and NH on
different polypeptide strands Strands can run
in same direction (parallel ?-sheet) or in
opposite direction (anti-parallel ?-sheet)
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Model of ?-sheet
Anti-parallel
SILK - anti-parallel ?-sheets Strong structure
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Special helix in collagen
Rod shaped structure - NOT same as ?-helix More
open than ?-helix 3 amino acid residues per
turn Conserved sequence Gly-X-Y-Gly-X-Y- (X and
Y are proline and hydroxyproline)
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Collagen triple helix
Each strand forms a helix 3 collagen strands
entwine - triple helix
Glycine occupies every 3rd position -
small, fits in interior ( Gly-X-Y-Gly-X-Y-Gly
-X-Y) Proline and hydroxyproline to exterior
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PROTEIN 3y STRUCTURE
Side chain (R group) interactions Most are
weak, non-covalent interactions - but
stabilise protein Spatial arrangement and
interactions of AAs far apart Responsible for
folded, biologically active protein
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3D STRUCTURE Of myoglobin
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TYPES OF BONDING
H bonding (ser, thr OH groups) Electrostatic
interactions between charged groups (lys, arg,
his, glu, asp) Hydrophobic interactions (leu,
val, phe.etc) Van der Waals forces (short
range) Disulphide bonding between cysteine
residues Strong interaction
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3y structure bonds
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PROTEIN DENATURATION
DISRUPTS BONDING insoluble Heat
- breaks weak bonding (eg egg white) pH
-affects H bonding and electrostatic
interactions Detergents and organic
solvents(hydrophobic bonds) Chaotropic agents
eg urea, guanidine HCl Form H bonds with AAs and
disrupt existing H bonding and hydrophobic
interactions
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4y STRUCTURE
Complex of 2 or more separate polypeptide
chains Interactions between subunits Non-covale
nt and covalent interactions
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MODEL of HAEMOGLOBIN 4 subunits interacting Has
4y structure
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SUMMARY OF PROTEIN STRUCTURE
1Y
3Y
4Y
2Y
Tetrameric protein
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Importance of protein structure
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3D STRUCTURE IMPORTANT FOR PROTEIN
FUNCTION
Allows protein to interact with other
molecules Form bonds with other
molecules Usually weak but specific
interactions Why proteins act as enzymes,
receptors etc
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IMPORTANCE OF 1y STRUCTURE
Denaturing agents Urea, guanidine HCl - disrupt
H and hydrophobic bonds Mercaptoethanol -
reducing agent Reduces disulphide bonds (-S-S-
- SH ) - SH
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Denaturation of 3y structure
Denatured protein
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Denaturation of 3y structure can be reversed
Remove urea and mercaptoethanol
Denatured protein
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CONCLUSION
All information for complex 3D structure
contained in amino acid sequence 1y sequence
dictates structure Chemically, folding slow
process Physiologically, protein folding
involves chaperone proteins - scaffold, help
folding
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IMPORTANCE OF CORRECT 1Y STRUCTURE
GENE MUTATIONS Changes in AA sequence SUBSTITUTIO
NS - conservative - same type of AA - radical
- different type AA Change can affect ligand
binding or enzyme activity Can affect shape of
protein (esp cysteine substitutions)
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INSERTIONS OR DELETIONS Can cause loss of
protein function But not always - if remove
non-essential part
Deletions/substitutions - can determine
binding site residues
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PROTEIN ENVIRONMENT
IN AQUEOUS ENVIRONMENT
e.g. water soluble protein HYDROPHILIC
AMINO ACIDS OUTSIDE (H bonding, electrostatic
interactions) HYDROPHOBIC AMINO ACIDS
INSIDE (hydrophobic interactions, Van der Waals
forces)
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Haemoglobin model
Hydrophilic
Hydrophobic
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IN HYDROPHOBIC ENVIRONMENT eg transmembrane
protein HYDROPHILIC AAs on AQUEOUS
SIDE HYDROPHOBIC AAs IN MEMBRANE LIPID BILAYER
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MODEL OF GLUCOSE TRANSPORTER
HYDROPHILIC
membrane bilayer
HYDROPHOBIC
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FIBROUS and GLOBULAR PROTEINS
FIBROUS Usually structural proteins eg
collagen, keratin, actin, myosin Extracellular or
intracellular Extensive 2y structure, stabilised
by disulphide and H bonds Undergo modifications
- increase stability eg collagen
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GLOBULAR PROTEINS Mostly non-structural
proteins Compact shape 3y structure important
to function Ligand binding sites eg
myoglobin, haemoglobin, albumin, enzymes,
receptor proteins
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GENETIC OR OLD AGE Defect in maintaining
globular form - protein
destabilised to fibrous form TRANSMISSIBLE
(PRIONS) Already formed fibrils act as template
globular fibrous
Rapid progression
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• SUMMARY
• 1.Protein structure dictated by amino acid
  sequence
• 2. X-ray crystallography - 3D structure
• 3. Folding dependent on environment
• 4. 3D structure important for function
• 5. Gene mutations can affect biological activity
• 6.Protein can exist in globular or fibrous form

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4Y STRUCTURE
Protein with gt1 subunit Subunits
interact Influence binding at each binding
site CONFORMATIONAL CHANGES WHEN LIGAND BINDS
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ALLOSTERIC EFFECTS
Tense form (T) - Low affinity for ligand Relaxed
form (R ) - High affinity for ligand Ligand
binds to T, increases affinity of other subunit
2 subunit protein
T
R
R
T
T
R
COMMUNICATION BETWEEN SUBUNITS
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REGULATION OF PROTEIN ACTIVITY
ALLOSTERIC REGULATORS BIND AT OTHER SITES -
ALTER AFFINITY FOR LIGAND Positive allosteric
regulators increase ligand affinity Negative
allosteric regulators decrease ligand affinity
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ALLOSTERIC REGULATION
Protein/enzyme activity can be changed Regulated
activity Physiologically important
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DNA Genetic material Made up of
deoxyribonucleotides Linked by phosphodiester
bonds DNA double helix
PROTEINS Structural (eg collagen) Functional (eg
enzymes) Made up of amino acids Linked by
peptide bonds Alpha helix Beta sheet Collagen
triple helix
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