PROTEIN

STRUCTURE AND FUNCTION OF

BIOMOLECULES

(WEEK 9-10)
 PROTEIN
I. DEFINITION
II. GENERAL FORMULA
III. CHARACTERISTICS
IV. CLASSIFICATIONS AND NUMENCLATURE
V. STRUCTURE ORGANIZATION
VI. REACTIONS
VII. ANALYSIS: Isolation and Purification
Spectrophotometri
Electrophoresis
Chromatography
Affinity-based Method
X-Ray Crystallography
Sequencing
Synthesis
I. DEFINITION

● Poly-amino acid-macromolecules (polypeptide)

II. GENERAL FORMULA

n
 III. CHARACTERISTICS OF PROTEIN MOLECULE

1. Primary component of cell/life (forming structure/shape, controlling the process

within the cell/life, etc.)
2. Macromolecule:
Insuline → MW 5700 ( 50 aa)
ATPase → MW 510.000 ( 510 aa)
Tobacco Mosaic Virus → MW  450 million (millions of aa)
3. Composed of 20 aa → connected each other in variety by peptide bond → forming
polypeptide
4. Limited structure stability (easy to undergo denaturation)
Reversible/ireversible
Factors : pH, T, solvent, solute, ionic strength, radiation, etc.
5. Some are reactive → aa building units contain reactive functional groups, such as: -
SH, -OH, NH2, -COOH, -OH, etc.
Reactions are selective and specific → specific side chain and characteristic
macromolecular shape/composition
6. Contain various chemical bonds (hidrogen, covalent, vdw, ionic, electrostatic,
disulfide, etc.)
 IV. CLASSIFICATION

4.1 Based on the source 4.1 Based on the location within the cell
Animal Plant Cytoplasm Nucleus
Microorganisms Membrane Etc.

4.3 Based on the shapes

Globular
→ More complex, mostly reactive (enzyme, antibody,
transport protein , etc.), folding match to its function,
i.e. myoglobin, sitochrome c, hemoglobin, hexokinase,
lactate dehidrogenase, etc.

Fibrous
→ anatomy & phisiology, structural, outer defence (outer skin, hair,
nail, horn, etc.). Examples: -keratine, -keratine, kolagen,
elastine, myosin, actin, etc.
4.4 Based on the molecular weight (MW)

Proteins MW
Insulin (bovine) 5,733
Cytochrome c (human) 13,000
Ribonuclease A (bovine pancreas) 13,700
Lysozyme (egg white) 13,930
Myoglobine (equine heart) 16,890
Chymotripsine (bovine pancreas) 21,600
Chymotripsinogen (bovine) 22,000
Hemoglobin (human) 64,500
Serum albumin (human) 68,500
Heksokinase (yeast) 102,000
Imunoglobulin G (human 145,000
RNA polymerase (E. coli) 450,000
Apolipoprotein B (human) 513,000
Glutamat dehydrogenase (bovine liver) 1,000,000

Gravitational based separation

→ centrifugation, chromatography, electrophoresis, etc.
4.5 Based on the solubility

Proteins pI
Pepsin  1.0
Egg albumin 4.6
Serum albumin 4.9
Urease 5.0
-Lactoglobulin 5.2
Hemoglobin 6.8
Myoglobin 7.0
Chymotrypsinogen 9.5
Cytochrome c 10.7
Lysozime 11.0

pI-based separation/purification
→ each protein dissolves optimally at a specific pH → pI
Precipitation(salting out)
Ex: albumin (+ water), globulin (- water, garam encer/pekat), glutelin (-
water, + aliquots acid/base ), glialdin/prolamin (- water, ethanol 100 %, +
ethanol 70-80 %), histon, protamin (+ water, base)
4.6 Based on the polarity → polarity/charge-based protein separation →
(electrophoresis, ion-exchange chromatography, etc.)

4.7 Based on the association with other non protein substances (nukleo-, lipo-,
gliko-, metalo-, kromo, sulfa-protein)

Class Prosthetic group Example

Lipoproteins Lipids 1-Lipoprotein of blood
Glycoproteins Carbohydrates Immunoglobulin G
Phosphoproteins Phosphate groups Casein of milk
Hemoproteins Heme (ironporphyrin) Hemoglobin
Flavoproteins Flavinnucleotides Succinate dehydrogenase
Metalloproteins Iron Ferritin
Zinc Alcoholdehydrogenase
Calcium Caldmodulin
Molydenium Dinitrogenase
Copper Plastocyanin
 4.8 Based on the biological functions
Enzyme (luciferin + ATP Storage (casein)
+ luciferase → light)

Transport (erythrocytes
hemoglobin → O2 transport) Ovalbumin → egg.
Regulatory (growth Ferritin → bakteria,
hormone  pituitary) plant & animal tissue)
Defence (ricin) Structural (fibroin)

snake venom, Keratin → hair,

antibody, fingernails & feathers
Contractile/motile fibrinogen, Resilin → insects
(tubulin → microtubule or + dynein thrombine wing)
→ flagella or cilia)
 4.9 Other (unclassified) proteins
Monellin → Protein (african plant)
→ intensively sweer taste → food sweetener
→ nonfattening
→ nontoxic
Antifreeze proteins → Blood plasma of some Antartic fish
→ protect blood from freezing
Attachment proteins:
→ microbial interaction (attachment to surfaces/host)
→ root surfaces (plant)
→ oral surfaces
→ epithelial surfaces (rumen)
→ fresh water systems Multifunctional proteins
→ marine environment → various biological funtions
→ soil and sediments → viral protein coat
Receptor/detector/reporter proteins
chemo outer & cytoplasmic membranes
effectors flagellum
counter clockwise → tumble

clockwise → run
 PROTEIN COAT OF VARIOUS VIRUS

Capsid DNA
Variola major (pox) (head)
Neck
Collar
Sheath T4

Tail Base
fiber plate

HIV Membrane
envelope
RNA
Polio

Envelope proteins Reverse transcriptase

PROTEIN COAT OF TMV

RNA Protein
subunit

Total length
= 130 turns
 V. STRUCTURE ORGANIZATION OF PROTEIN (1)

● Primary structure Peptide bond

• peptide bond O O O O
• aa sequence H 2N CH C HN CH C HN CH C HN CH C OH
→ R sequence R1 R2 R3 Ri
→ folding
→ properties

● Secondary structure backbone folding

-plated sheet -heliks
or

● Quartenary structure
its association with:

- other substances → holoprotein ● Tertiary structure

- other protein → polisubunit protein (dimer,tetramer, etc.) polypeptide folding
- other substances and protein → complex protein  R sequence
V. STRUCTURE ORGANIZATION OF PROTEIN (2)

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 5.1 PRIMARY STRUCTURE

Peptide bond in polypeptide of

primary structure

Peptide bond
 5.2 SECONDARY STRUCTURE

• Backbone folding
• Supporting factors:
• Primary structure supports (peptide bond)
• Resonance of peptide bond (50% single and 50 % double bond)
→ limited rotation
-
+

H
100% single bond - 100% double bond
→ full rotation → no rotation
+
H

d-
d+ 50% single and 50 %
double bond
H → limited rotation

• Repeated hydrogen bonding between O of C=O and H of NH

• Repeated hydrogen bonding between O of C=O and H of NH
a. within the same polypeptide (intramolecular) →  helix

aa1 aa2 aa3 aa4 aa5 aa6

Bonding may form between aa1 and aa3 (2 M ribbon); aa4 (310 helix); aa5 (-
helix); or aa6 (-helix) but not with aa2 → unstable → too much energy required

 
helix pleated
sheet

b. between different polypeptide (intermolecular) →  pleated sheet

H H O O H H H H O H H O O H H HH O
H H C H H C
C N C C N C N H C C N C C N C N H C
N C C N C C N C C N C C N C C N C C
R1 R3 R5 R1 R3 R5
H O R2 H O R4 H O R6 H O R2 H O R4 H O R6

O H H H H O O H H H H O O
H H HH O
C H H H H C
C H N C N C C N C C N C C N C N H C
C C N C C N C C N N C C N C C N C C
R5 R3 R1 R1 R3 R5
R6 O H R4 O H R2 O H H O R2 H O R4 H O R6

Antiparalel ⇄ Paralel ⇉
 5.3 TERTIARY STRUCTURE
• Polypeptide folding due to the interactions of aa-R groups within the same polypeptide
chain (intramolecular)

H-N-R
H
a b
a O
-R-C-OH

c -R-
-R-
d
e

• Supporting factors:
• Secondary structure supports
• R groups interactions:
a. hydrogen bonding between -C=O and –NH or –OH)
b. ionicelectrostatic/polar hidrophilic interaction
c. non polar/hidrophobic interaction
d. van der Waals interaction
e. disulfide bond
 DENATURATION/RENATURATION OF TERTIARY STRUCTURE

Native ribonuclease Denatured ribonuclease Native ribonuclease

(enzymatically active) (inactive) (activity regained)

Denaturing Removal of
agents denaturing
agents

Disulfide bond Disulfide bond

 THE IMPORTANCE OF PROTEIN FOLDING (TERTIARY STRUCTURE)

● Polarity
● Solubility
● Position of functional
groups 2.5 nm
● Shape

Subsequent interaction
mixture of secondary structure in the tertiary protein structure

Unfolded polypeptide

Folded conformation in aqeous environment

 5.4 QUARTENARY STRUCTURE
• Interaction of protein (tertiary structure) with other substances:
• Non protein → holoprotein → → metal (metaloprotein), chrome (chromoprotein), lipid
(lipoprotein), carbohydrate (glycoprotein),etc.
• Other protein → polysubunit protein → dimer, tetramer, etc.
• Protein & non protein → complexes protein

3o, monomer 4o, dimer

4o, monomer
4o, tetramer

Quartenary structure
4o, dimer (4 monomers combine to form hemoglobin)

Cross
section
of hair
Cells
• Supporting factors:
• Tertiary structure supports Filament
• Interaction between subunit (hydrogen, ionic, polar, non polar, Two-chain coiled coil
disulfide, etc., or other bonds that have not been used in tertiary -helix
structure) and other subunit or other non protein molecules
 VI. REACTIONS WITH PROTEIN
Coloring reactions
Millon ↴ white → red
Hopkins Cole → violet ring at the interface
Biuret Cu2+ → complex of Cu2+-peptida + Na-wolframate
→
Lowry (phosphomolibdate) → green → blue
Xanthoprotein → yellow → orange (base) → aromatic: trp, tyr, phe
Ninhidrin → 1 carboxilate & 1 free amino → blue
Reaction with metal → -SH or negative charge
Denaturation/renaturation: → reversible (r) → weak bond
→ ireversible (i) → strong (covalent) bond
temperature → (i) → destroy weak (not too hot) and strong (hot) interactions
pH → weak acid/base → (r)
→ strong acid/base → (i) → destroy charge balance → resulting in
electrostatic repulsion and hydrogen bonding disruption
organic solvent (alcohol, acetone, etc.) → (i) → interferes the solubility
solute → urea, detergen (i) → interferes hydrophobic interactions that
stabilizes globular protein
Hidrolysis: acid (HCl, TCA, etc.); base (NaOH); enzim (specific for peptide bond
near certain aa)
Precipitation: etanol, aseton, or urea
 ACID/BASE DENAT URATION
Acids and Bases Disrupt Salt Bridges
• Salt bridges result from the neutralization of an acid and amine on side chains. The
final interaction is ionic between the positive ammonium group and the negative acid
group.
• Any combination of the various acidic or amine amino acid side chains will have this
effect.
• Acids and bases disrupt salt bridges held together by ionic charges. A type of double
replacement reaction occurs where the positive and negative ions in the salt change
partners with the positive and negative ions in the new acid or base added

Tertiary Structure-Salt Bridges

+ + Acid +
glutamate lysine
 HEAVY METAL DENAT URATION
• Heavy metal salts act to
denature proteins in much
the same manner as acids
and bases.
• Heavy metal salts usually
contain Hg+2, Pb+2, Ag+1
Tl+1, Cd+2 and other metals
with high atomic weights.
Since salts are ionic they
disrupt salt bridges in
proteins. The reaction of a
heavy metal salt with a
protein usually leads to an
insoluble metal protein
salt.
• Heavy metals may also
disrupt disulfide bonds
because of their high
affinity and attraction for
sulfur and will also lead to
the denaturation of
proteins.
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 ALCOHOL DENATURATION
• Alcohol Disrupts Hydrogen Bonding
• Hydrogen bonding occurs between amide groups in the secondary
protein structure. Hydrogen bonding between "side chains" occurs in
tertiary protein structure in a variety of amino acid combinations.

Tertiary Structure-Hydrogen Bonding

asp tyr

Denaturation by Alcohol

H
asp O-CH2CH3

CH3CH2-O
H tyr
 VII. PROTEIN ANALYSIS
7.1 Isolation of protein
7.1.1 Presipitation
8.1.1.1 Organic solvents (ethanol,
acetone,etec.)
8.1.1.2 Solutes (urea, detergent, etc.)
7.1.2 Gradient centrifugation Sucrose gradient Sample in
sucrose gradient
7.1.3 Advance analysis Centrifuge at high speed
→ (further purification)
→ electrophoresis, chromatography,
affinity, etc.
Separated
macromolecules
7.2 Concentration determination (spectrophotometri) or organelles
7.2.1 Hydrolytic method
Protein → aa → protein estimation based on spesific aa content  with
(ninhidrin, fluoroscamin, etc.) or without (based on trp or tyr) addition of
spesific reagents → method for analysis
7.2.2 Non hydrolytic method
Protein + spesific reagent → (Millons, Hopkins Cole, Biuret, Lowry, Bradford,
Xanthoprotein, etc.) → spectrophotometry analysis
7.2.3 More advance analysis
→ (further purification) → electrophoresis, chromatography, affinity, etc.
 7.3 PROTEIN ELECTROPHORESIS (1)
7.3.1 SDS-PAGE → MW 7.3.3 IEF → pI
- MW>>
pH = 9 - pI>>

+ pI<<
+ MW<< pH = 3
7.3.4 Two dimensional electrophoresis
7.3.2 Determination of MW
First
dimension
(IEF)
pI<<
Second
dimension
(SDS-PAGE)

Two
dimensional
gel Mr<<
pI<<
7.3 PROTEIN ELECTROPHORESIS (2)
 7.4 CHROMATOGRAPHY: TYPE OF CHROMATOGRAPHY
SIZE HYDROPHOBICITY CHARGE BIORECOGNI- HYDROPHOBICITY
TION (LIGAND
SPECIFICITY)

GEL HYDROPHOBIC ION REVERSED

FILTRATION INTERACTION EXCHANGE AFFINITY PHASE
GENERAL TERMS IN CHROMATOGRAPHY
ELUTION METHODS
 7.4 CHROMATOGRAPHY: 7.4.1 HPLC

• Low affinity for Mobile phase

stationary phase Molecules to be separated
• Medium affinity for
stationary phase Stationary phase
• High affinity for Column
stationary phase

Solution moves down

column through
stationary phase
Chromatogram
Absorbance

Molecules with
0 20 25 30 lowest activity for
Time (min) stationary phase
emerge first

Detector
 COLUMN CHROMATOGRAPHY
Protein separation based on the interaction of protein with stationary phase
 7.4.2 PARTITION CHROMATOGRAPHY

Porous
polymer
beads

Protein mixture is added

to column containing
Chromatogram cross-linked polymer
Absorbance

Detector Protein molecules separate

by size: larger molecules
Time passmore freely,appearing in
the earlier fractions
 7.4.3 ION-EXCHANGE CHROMATOGRAPHY
Reservoir of buffer allows
sample to percolate slowly
through column
+

Solutions of amino acids at

pH 3.0 is poured onto a
cation-exchange column

Amino acids with greatest

positive charge (red) bind the
coloumn most tightly and
therefore move most slowly.
Those with the least amount
of positive charge (blue)
move fastest and elute first

Chromatogram
Absorbance

Fractions are collected

Detector from the bottom of the
column and analyzed
quantitatively
Time (minute)
7.4.3 ION-EXCHANGE CHROMATOGRAPHY
 7.4.4 AFFINITY CHROMATOGRAPHY (1)

Protein mixture is added to the column containing a

polymer-bound ligand spesific for protein of interest

Solution
of ligand

Unwanted proteins Protein of interest

are washed trough is eluted by ligand
column solution
= Ligand = Protein of interest
Chromatogram
Absorbance

= ligand coupled to polymer bead Detector

Time
 7.5 AFFINITY ANALYSIS: 7.5.1 ELISA
● Indirect Antigen ● Direct
Antibody
1. Antigen is bound to microfilter
1. Antibody to antigen us bound to
microfilter well
Antibody
for serum Antigen
2. Antibody from serum added, wash
2. Blood sample added as a source of antigen
Open-
faced Double
sandwitch Secondary antibody Secondary
Antibody sandwitch Antibody
3. Anti-human immunoglobulin antibody 3. Antibody labeled with enzyme (E) added.
labeled with enzyme (E) added wash Enzyme labeled antibody binds to antigen

4. Add enzyme substrate (S). Colour formed 4. Substrate (S) for enzyme is added. Colour
(P) is proportional to amount of antibody product of enzyme (P) is proportional to
in serum amount of antigen
Colour Colour
Standard curves
Antibody Antigen
 7.5.2 RIA
STANDARD CURVE Insulin Microtiter wells
1 2 3 4
Add excess anti- Increasing concentration
insulin antibodies that Radioactive antibody
are labeled with
125Iodine; wash to
remove unbound
antibody Radioactivity
Count radioactivity in
gamma radiation
counter to establish a
standard curve with
known amounts of Insulin
1 2 3 4
antigen (insulin)
Subject A Subject B Subject C Subject D
SAMPLES
Radioactivity
Proceed samples as Subject B, D CONCLUSION:
above and plot the A: diabetic
results to the standard Subject C B, D: normal
curve C: potentially diabetic
Subject A
Insulin
1 2 3 4
Insulin
 7.5.3 Immunobloting

• Immunobloting
7.5.4 CYTOCHEMISTRY STAINING
7.6 X-RAY CRYSTALLOGRAPHY

ANIMATED X-RAY
CRYSTALLOGRAPHY
 VIII. PROTEIN/PEPTIDE SEQUENCHING
• 8.1 Breaking disulfide bonds Disulfide bond
(cystine)

Cysteic acid residues

• 8.2 Cleaving the polypeptide chains Acetylation by iodoacetate
• 8.3 Sequenching of peptides
• 8.4 Ordering peptide fragments
• 8.5 Locating disulfide bonds Acetylated cysteine residues

protein → breaking disulfide bonds → cleaving

the polypeptide chains (specific enzyme)

electrophoresis (compare the patterns)

protein → cleaving the polypeptide chains (specific enzyme)

 7.7.3. Peptide Sequenching (Edman method/degradation)

H
Phenilthioisocyanate + H-N-aa1-aa2--------- COOH

HN-aa1-aa2---------- COOH

H2N-aa2--------- COOH HN-aa1

Identify
 7.7.3. Peptide Sequenching (Edman method/degradation)

S
-N=C +
Phenylisothiocyanate
1 OH- (mildly alkaline condition)
Phenylthiocarbamoyl
(PTC)
-N=C
adduct
S

Anilinothia- 2 CF3COOH (anhydrous trifluoroacetic acid)

zolinone-aa

Phenylthio- O
hydantoin-aa R1 3
C CH Organic extraction and
N aqueous acid treatment
Purify and recycle
C NH
Identify aa through step 1-4
S 4
 Sanger reagent

Edman degradation

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8.3. Peptide Sequenching (Edman Method/Degradation)
7.7.4 Ordering Peptide Fragments
 7.8. PROTEIN/PEPTIDE SYNTHESIS
Insoluble polystyrene bead
α-Amino group protected by
t-butyloxycarbonyl
Cl- amino
CF3COOH
3. Amino acid with
protected α-amino
group is activated
Dicyclohexylcarbodiimide at carboxylgroup
by DCC
HF
1. Attachment of
carboxyl terminal
acid to
reactive group on
resin
2. Protecting group is
removed by flushing
with CF3COOH
4. α-Aminogroup of
amino acid 1 attacks
activated carboxyl
group of amino acid 3
to form peptide bound
Dicyclohexylurea
Reactions 2 to 4
repeated as necessary
5. Completed peptide is
deprotected as in
reaction 3. HF
hydrolyzes ester
linkage between
peptide and resin
 OLIGONUCLEOTIDE SYNTHESIS

Nucleotide
activated at
Repeat steps 2 3’ hydroxyl
to 4 until all
residues are 4. Oxidation to
added form triester
3. Next nucleotide added

5. Remove blocking groups from bases 5’ 3’

6. Remove methyl groups from phosphates
7. Cleave the chain from silica support Oligonucleotide chain

1. A nucleotide attached 2. Protecting group

to silica support (Si) (DMT) removed
Nucleotide protected
(DMT) at 5’ hydroxyl