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DR. M. Sasvári
Biochemistry Lectures
Amino Acids, Proteins, Enzymes
Proteins
Proteins are polypeptides
Polypeptides: The peptide bond
Peptide bond: Condensation reaction
between the -carboxyl group and the -amino group:
-H2O
The peptide group is rigid and planar
carbonyl group
O
C
p bond
RESONANCE INTERACTION
..
N
lone electron pair
of N
H
C-N bond will have some double bond character
Consequences:
1. Planarity
2. Trans conformation
Trans configuration of the peptide group
How to write a dipeptide?
C-terminal
N terminal
+H
3N
H
O
R
C
C
C
N
R
H
H
COO-
A biologically important tripeptide: Glutathion
+
Glu-COOH
Glu-Cys
ATP
H2N-Cys
-glutamyl-cystein
-glutamyl-cystein synthetase
ATP
H2N-Gly
CH2SH
O
3N
CH 2
C  - COO
H
N
H
H
N
C
C
O
+H
H
C
C
H2C 
-glutamyl-cysteinyl-glycine
glutathion
glutathion synthetase
H
+
Glu-Cys
Glu -Cys-Gly
H
COO
-
Function of Glutathion
2 G-SH + H2O2
G-S-S-G + 2H2O
reduced glutathion (thiol group)
oxidized glutathion (disulphide bridge)
Importance: Maintaining of cellular redox state
Reductive power against oxidative stress
Red blood cells: high O2 concentration
oxidative effect
free radical formation
hydrogen peroxide formation
lipid peroxidation
NutraSweet® (aspartame), an artificial sweetener
Dipeptide of Asp and Phe (carboxyl terminal is esterified)
C-terminal
N terminal
+H
3N
H
O
CH2
C
C
C
CH2COO
N
-
H
CO-O- CH3
methylester
H
Polypeptides: Isoelectric point
Example:
Glu-His-Arg-Gly
Sequence of a tetrapeptide:
Charges at pH 7:
Isoelectric form:
Calculation of Ip:
+
+
-
+
+ Arg (guanidino)
+ -amino group
+ His (imidasol)
-
-
(pKa = 12.5)
(pKa = 9.7) Next
(pKa = 6.0) Lost
Glu-carboxyl group)
-carboxyl group
Ip:basic
+
+
(pKa = 4.3)
(pKa = 2.3)
Ip: (6.0 + 9.7)/2
Other biologically important peptides
Peptide hormones:
thyrotropin releasing factor (3 amino acid residues)
oxytocin (9) – uterin contraction
bradykinin (9) – inhibits inflammation
enkephalins (CNS)
small proteins – large peptides
insuline (30+21)
glucagon (29)
Levels of protein
structure
Main molecular forces
Primary structure
- Covalent bond (peptide bond and disulphide bridge)
Secondary structure
- H-bonds between the atoms of the peptide group
Tertiary structure
- Secondary bonds between the side chains of the same
polypeptide
Quaternary structure
- Secondary bonds between the side chains of different
polypeptides
Primary structure
- Amino acid sequence
Secondary structure
- -helix, b-sheets, collagen helix, random coil
Tertiary structure
- Conformation
Quaternary structure
- subunits
Primary structure: The amino acid sequence.
The primary structure is defined by the
covalent bonds that hold the molecule together.
- peptide bonds
- disulfide bridges
for example Bradykinin : Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
Hydrolysis of proteins: covalent bonds
(primary structure) are broken
Peptide bonds can be cut by hydrolysis
Chemical:
cooking the proteins in strongly basic solutions
result: mixture of amino acids
Enzymatic:
proteases (endopeptidases)
result: smaller peptides
-CO-NHpeptide1
peptide2
+ H 2O
The peptide bond is rigid and planar
Six atoms of peptide group: C1 – CO-NH-C2
Limited rotation of atoms around the peptide bond
Rotation is permitted at the N-C (f) and C- C () bonds
Secondary structure
Conformation of the polypeptide backbone
The conformational relationship of neighboring amino acids :
-helices, b-plated sheets, or "random coils"
Molecular forces:
H-bond between the atoms of the peptide groups
H
C
N
Backbone structure
Side chains has only small influence
O
H
C
O
N
Examples:
-helix
parallel and antiparallel b-sheets
collagen helix (see later)
Ramachandran plot:
O
C N
H
R
f
C
H

prediction of secondary structure
O
C N
H
possible angles of free rotation
b strands
 helix
Forces of the secondary structure
Stability of the -helix and b-sheet is based on
- the minimized steric repulsion between the side chains
- the maximized H-bonds between the peptide groups
Stability of an -helix is decrease by
1. electrostatic repulsion (or attraction)
of adjacent positive (or negative) side chains
2. bulkiness of adjacent side chains
3. (ionic) interaction between amino acid side chains
spaced tree (four) residues apart
4. positively charged amino acid at the C-terminal, or
negatively charged amino acids at the N terminal
5. presence of Proline
Right handed -helix
http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG04_10.JPG
Left
Right
handed helix
-helix, Space-filling models
view from one end
looking down the longitudinal axis
R groups protrude outward
from the helical backbone
Secondary structure of proteins
b-sheets: extended, zizag structure
Antiparallel chains of b-conformation
H-bond
Alternating pattern of R groups
Other secondary structures
b-turn
A connection between
the ends of antiparallel b-chains
• a tight connection
• involves 4 amino acid residues
• H bond between the peptide groups
of the 1st and 4th amino acids
• Gly and Pro occur frequently
• often found near to the surface of the protein
Protein folding: Tertiary structure
Amino acid side chain interactions of the same polypeptide chain
Longer-range aspects of amino acid sequence
Types of interactions:
nonpolar (Wan der Waals)
polar
H bonds
ionic interactions
hydrophilic surface
Polar interaction
of side chains with water
hydrophobic
pocket
of a water soluble
protein
hydrophobic interactions
between
hydrophobic side chains
Conformations from many helices
Conformations from bsheets
Quaternary structure – subunit structure
Amino acid side chain interactions of different polypeptide chains
Longer-range aspects of amino acid sequence
Types of interactions:
nonpolar
polar
H bonds
ionic interactions
A leucin zipper (DNA binding protein)
2 polypeptides – 2 subunits
Quaternary structure – subunit structure
HIV-1 aspartic protease
Chicken triose phosphate isomerase
2 identical all-beta subunit
2 identical subunits
with alpha/beta barrel folds
Myoglobin
and
Hemoglobin
Myoglobin
Heme
• Extremely compact
• 75% -helix, 8 helices
• 4 terminated by Proline
C
• 5 nonhelical segments
and two additional
non-helical regions:
• C terminal: HC1 - HC5
• N terminal: NA1 & NA2
Nonpolar side chains inside
(except two His)
Heme is in a crevice, inside
(nonpolar surrounding)
Polar side chains outside
distal His
proximal His
N
Myoglobin
Model of the
oxygen binding site
Fe2+:
• 6 coordination positions
• 4 occupied by the heme
• 1 by proximal His
• 1 by O2
Fe3+: binds H2O
O2 binding :
bent mode (angle)
121 O
Functions of His
Proximal His:
holds the heme
Distal His:
bent mode binding of O2

weakens CO binding
(endogenous CO production
occupy 1% of sites)
Comparison
Myoglobin
monomer
Hemoglobin
tetramer
allostery (T and R forms)
homotrope cooperativity
BPG binding
Hemoglobin: a tetramer (12b1b2)
b subunit has a high homology to myoglobin
Homotrop cooperativity between the subunits
high O2 pressure
saturated with O2
R forms
O2
T/R
low O2 pressure
BPG
O2
T forms
O2
O2
BPG has negative charges - ionic bonds
BPG binds to the center of Hemoglobin
O2 saturation curves of different forms
High-affinity
state
Transition
from
low- to highaffinity state
Low affinity
state
O2 saturation curve of Hemoglobin
without BPG
similar to
myoglobin
with BPG
sigmoid curve
(cooperativity)
Hemoglobin: allosteric forms
Relaxed (R ) form
Tense (T) form
binds O2
releases O2
no BPG
BPG
secondary bonds
ionic bonds
lung (high O2 pressure)
tissue (low O2 pressure)
Triggering the T  R conformational change
Helix F
Deoxyhemoglobin (T form)
Fe2+ is out of the plane of the heme

protrude His F8, and the F8 helix
Helix F
Oxyhemoglobin (R form)
O2 binds to heme  heme becomes planar

initiation of movement
(His F8 and +F helix)
T form has ionic bonds  broken in R form
e.g. T form: His HC3 - Asp F/G1
Conformational changes during T  R transition
Rotation of His HC3residue
T form
R form
- ionic interactions are broken
- pocket is narrowed - no place for BPG
Structural explanation of Bohr effect
R forms
strongly binds O2
BPG
O2
O2
T forms
stabilized by BPG
O2
O2
Abnormal
Hemoglobins
Subunits of human hemoglobin
Adult Hgb
Hb A
Hb A2
2 b 2
2 d2
Small differences in amino acid sequence
Amino acids in BPG binding:
His 143
His2
-amino terminal
Lys
Fetal Hb
Hb F
22
His 143  Ser 143
weaker BPG binding  higher affinity to O2
Abnormal hemoglobins
More than 300 allele/subunit
- neutral polymorphism
- harmful mutations
HgM and HgS
Hg M
proximal His  Tyr
Tyr - O Water binds instead of O2  lethal
Hgb S
Glu6  Val6
Fe3+
H2O
Tendency for aggregation in deoxyHb
Sickle cell trait
Heterozygotes : 50% HbS - 50% HgA
About 1 % of RBC are sickled
Resistance to malaria
Balanced polymorphism
Homozygote: lethal
Prenatal diagnosis (chorion villi sample)
Thalassemias: defective synthesis of Hb
Hydrophobic patch on deoxyhemoglobine in Hgb S
Polymer formation
in Hgb S
DR. M. Sasvári
Biochemistry Lectures
Amino Acids, Proteins, Enzymes
Fibrous Proteins
-Keratin, Silk Fibroin, Collagen, Elastin
-Keratin:A fibrous protein
•hair
•nails
•outer layer of skin
-helical structure (Nonpolar amino acids, no Pro)
Protofibril
2 right handed  -helices:
2 -helices
a left handed double-strand
Non helical
regions
2 double stranded helices:
Protofibril
(2 coiled coils)
a left handed supercoil
Microfibril
Protofibril
8 protofibrils
arranged as a hollow circle
8 protofibrils
arranged as a hollow square
.
Nonpolar side chain interaction
S-S bridges
• A few -S-S- bridges (wool):
soft, can be stretched
• Many -S-S- bridges (claw):
hard and rigid
Cross section
of the hair
Cells
Filaments
Two chain
coiled coil
-helix
Curly and straight hair, permanent waiving
S-S
S-S
S-S
S-S
S-S
S-S
SH HS
SH HS
Red
SH HS
SH HS
SH HS
SH HS
CURL
Ox
Silk fibroin
Produced by silkworm
A fibrous protein
Primary structure:
Secondary structure:
Repeated sequence
(Ser-Gly-Ala-Gly)n
Antiparallel b-plated sheets
(Mw:  400 000/ polypeptide)
Tertiary structure:
Stacked b-sheets
H -bonds, nonpolar
interactions
Structure of silk fibrion, ball-and-stick model.
Antiparalell b-plated sheet
Collagen: a fibrous protein
Primary structure:
•tendons
•cartilage
•bones
Repeated sequence: (Gly-X-Y)n
X,Y: mostly Pro and HyPro
Post-translational modification
CO
CO
Oxidative decarboxylation
with hydroxylation
N
HN
4
Vit C
- HyPro -
- Pro -ketoglutarate + O2
Succinate + CO2
Other hydroxylation: Lys  5-hydroxyl-Lys
Vitamin C deficiency (Scurvy) : Unproper hydroxylation
OH
Secondary structure
Left handed helical structure
1 polypeptide
Gly
Pro
Hypro
3 residues per turn
Secondary structure
Left handed helical structure
1 polypeptide
Gly
Pro
Hypro
3 residues per turn
Tropocollagen: Right handed triple helix
from 3 left handed collagen helix
Stabilizing forces:
1. Interchain H- bonds
between peptide groups
- Gly -
- Pro -
2. Cross links between 2 Lys ( no Cys)
Crosslink between 2 Lys
- CH2- CH2 - CH2 - CH2 - NH2
Lys residue
O2
Oxidative deamination
NH3 + H2O
O
- CH2- CH2 - CH2 - C
H
+ H2N- CH2 - CH2 - CH2 - CH2- H2O
Lys residue
- CH2- CH2 - CH2 - C
N - CH2 - CH2 - CH2 - CH2 Shiff base linking two polypeptides
Reduction
- CH2- CH2 - CH2 - CH2-HN - CH2 - CH2 - CH2 - CH2 Lysinonorleucin bridge
Procollagen:
Globular parts at the ends
Function of
globular parts :
1. Promote
folding
2. Inhibit
premature fiber formation
Inherited disease:
Osteogenesis
Imperfecta
Gly Cys
988 988
Folding is incomplete
Procollagen is secreted from fibroblasts
Procollagene peptidase ( extracellular space) tropocollagen
Tropocollagen polymerization, crosslinkeage (Lys-Lys)
0.35 nm
Ehler-Danlos syndrome (strechable skin, hypermobyl joints)
Decreased activity of procollagen peptidase
DR. M. Sasvári
Biochemistry Lectures
Amino Acids, Proteins, Enzymes
Analysis and separation
of proteins
Gel electrophoresis (practice)
SDS-PAGE
isoelectric focusing
two-dimensional electrophoresis
Gel filtration (practice)
Dialysis (desalting)
Affinity chromatography
Ion exchange chromatography
Analysis of protein mixtures – SDS-PAGE
1. SDS-PAGE (see: practice book) – separation of protein mixtures
Denaturation of proteins, negative charges
SDS-PAGE: Cotrolling the purification procedure
Crude extract (1st lane)
Proteins after the purification steps (others)
Purified protein
four subunits (after denaturation)
SDS-PAGE: Determination of molecular weight of proteins
Analysis of protein mixtures: Isoelectric focusing
Analysis of protein mixtures: Two dimensional electrophoresis
1st dimension:
Isoelectric focusing
2nd dimension:
SDS-PAGE
Identification of more than 1000 proteins from E. Coli
Dialysis: Separation of proteins from small molecules (desalting)
Gel filtration: Separation of proteins from small molecules (desalting)
Protein purification: Affinity chromatography
Separation is based on the
specific binding
between the LIGAND and the protein
The ligand is cross-linked to the beads
Protein purification: Ion exchange chromatography
Separation is based on
charges of the proteins
(could be used for amino acids, too)
Chromatography
paper ~ (see prctice)
thin-layer ~ (see prctice)
column ~