Chapter 3

PROTEIN

 Amino acids
 Protein Structure (including Fibrous and Globular Protein)
 Protein Purification
 Nitrogen Metabolism
 Enzyme and Enzyme Kinetics

Learning outcome:
 Understand the structures and properties of amino acids
 Able to identify amino acids polarity and structure
 Able to explain the structure of primary, secondary, tertiary and quaternary structure of protein
 Understand the protein extractions procedures
 Able to differentiate different type of types of chromatography for protein purifications
 Understand how is nitrogen from the atmosphere can be turn into useful compounds
 Amino Acid Biosynthesis :Understand amino acid anabolic process through transamination and
one carbon transfer
 Understand what is essential and non essential amino acids
 Amino Acid Catabolism: Able to identify the fate of amino acid breakdown to amino group (to
urea cycle) and carbon skeleton (ketogenic and glucogenic pathway)
 Explain enzyme, substrate and enzyme specificity
 Able to explain factors affecting rate of enzymatic reaction
 Understand the function of the non-protein substance in enzymatic reaction and give examples
 Understand the enzyme kinetics involving Michaelis-Menten equation and Lineweaver-Burke
plot

Introduction

1. It is important to learn the structure and chemistry of the amino acids and other building blocks of
biological molecule to understand the fundamentals of proteins and enzymes, or the nucleic acids.
2. 20% of the human body is made of proteins. A large portion of our cells, muscles and tissue is
made of amino acid, meaning they carry out many important bodily functions, such as giving
cells their structure.
3. Amino acids have an influence on the function of organs, glands, tendons and arteries. They are
furthermore essential for removal of all kinds of waste deposits produced in connection with the
metabolism.
4. Other importance of protein, are producing:
 Hormones
 Enzyme
 Genetic sequence in nucleic aicd (DNA and RNA)
 Fiber muscles
 Immunoglobins for immune system
Amino acids Structure and Properties
1. Amino acid is an organic compound consists of
central tetrahedral carbon atom (α-carbon) that is
attached to amine group, carboxyl group and R-
side chain. The R-side chain determines the
different amino acids
2. Amino acid is a monomer of protein.

3. There are 20 common amino acids:

Acidic: If the R group contains carboxylic acid Basic: If the R group contains an amine group
 Lysine
  Histidine
  arginine

Polar (Hydrophilic): If the R group contains acids, Non-Polar (Hydrophobic): If the R group doesn't
bases, amines or alcohols contain any acids, bases, amines or alcohols.
 Aspargine  Threonine  Leucine  Methionine
 Serine  Tryrosine  Alanine  Phenylalanine
 Glutamine  Cysteine  Glycine  Tryptophan
 Proline  Isoleucine
 Valine

4. Named 2 amino acids for each:

a. Aromatic amino acid :

b. Sulphur-containing amino acid:

5. Amino acids can join via peptide bonds (amide bond) to form polypeptide chain (protein)
Amine group would attack the carbonyl carbon, removing H20 and forming CO-NH (amide
bond) .
Exercise:

Label the N- terminus and C-terminus in the polypeptide chain. And draw the formation of
peptide bonds between the two amino acids.

+ →

6. The peptide bond is very difficult to hydrolyze. It requires a strong base, or a biological enzyme
to break the peptide bond
7. Amino acids in protein:
a) L- amino acids are found in most protein
b) D- amino acids occurred in bacterial cell wall and antibiotics
8. Other type of amino acids that can be found :
a) Hydroxylysine, hydroxyproline – collagen
b) Carboxyglutamate - blood-clotting proteins
c) Pyroglutamate – in protein
d) Phosphorylated amino acids – a signaling device, enzyme inhibitor

9. 3 D structure of amino acids is important to observe chirality of amino acid.

Position of the OH/ amino group will determine D and L configuration for Fisher projection
Exercise:
Label the chiral carbon, and the D and L configuration in the amino acid diagram below:

pH scale
1. Amino group (+ ly charged)- proton acceptor , makes it more acidic
2. Carboxyl group (-vely charged)- proton donor , makes it acidic
3. There is an internal transfer of a hydrogen ion from the -COOH group to the -NH2 group to leave
an ion with both a negative charge and a positive charge, resulting zero net charge amino acids
called zwitterions.

4. The point on the pH scale that we can find the zwitterion is what we call the isoelectric point
5. Isoelectric point is the point along the pH scale where the molecule (amino acid) exist in a neutral
form with a zero net charge
6. Why is it important to know this?
So we can predict the charge of the amino acid at a certain pH. and predict the general structure
of amino acid
7. In that case, what happens when an amino acid is placed in acidic solution (lots of H+)?
The amino group will accept the excess H+ and form positively charged NH3 –
The carboxyl group will also gain proton and become uncharged
Thus the amino acid will be positively charged
 8. What happens when an amino acid is placed in basic solution (lots of OH-)?
The amino group will be deprotonated the excess OH- and form NH2 – no charged
The carboxyl group will also be deprotonated and become negatively charged
Thus the amino acid will be negatively charged

9. Spectroscopic Properties of amino acids

 All amino acids absorb infrared (IR) wavelengths
 Only Phe, Tyr, and Trp absorb UV
 Absorbance at 280 nm is a good diagnostic device for amino acids – useful for detection
of proteins contamination in DNA extraction
 Many chromatographic methods exist for separation of amino acid mixtures
a) Ion exchange chromatography
b) High-performance liquid chromatography

Protein Structure
1. There are 4 levels of protein structure:
a) Primary Structure
b) Secondary Structure
c) Tertiary Structure
d) Quaternary Structure

2. Primary Structure
a) A polymer of amino acids bind covalently by peptide bonds a.k.a polypeptide chain
b) Starts from the N-terminal end to the C-terminal end
c) Primary structure is more prominent compare to all other protein levels because the amino
acids sequence will determines the 3D structure, in return determine the properties and
protein function
d) A single amino acid substitution can give rise to a malfunctioning protein as in the case with
sickle cell anemia.Change in 1 amino acid residue in the sequence caused the RBC to unable
to bind to O2 effectively
3. Secondary Structure
a) A polypeptide chain arranged in 3-D structures which are strengthened by H-bond.
b) The 2° structure is maintained by hydrogen bonds between H of the N (amide hydrogen) and
the O of C=O (carbonyl oxygen).
c) Produce 2 shape of 3D structures:
a. α-helix sheet
b. β-pleated sheet
c. Sometimes may contain both shapes in one structure

α-helix

• a helical shape of one polypeptide chain

How is it stable?
• The helical conformation allows linear
arrangement of every atoms involved in H-bond,
giving the bonds its max strength and the helical
structure more stable

Factors that may disrupt α-helix :

• Rotation around the bond between the Nitrogen of
the α-carbon is severely restricted
• Proline’s presence can cause a chain to bend
because it cannot from intrachain with H-bond
• In α-helix structure, all side chain lies outside the
structure, therefore may caused crowding

β sheets
• When two polypeptide arrange adjacent to each
other , they can form bridges by H- bonds.
• C=O and N-H groups of each peptide bond are
perpendicular to axis of the sheet
• Less stable than α-helix
• There are two orientation to form a β-sheet
a) parallel or;
b) anti-parallel
• The orientation effects the pattern of H- bonds and
the general structure and function of the protein
• Stability : Anti parallel > Parallel

d) Supersecondary structures: the combination of α and β sections, as for example :

– βαβ unit: two parallel strands of β-sheet connected by a stretch of α-helix
– αα unit: two antiparallel α-helices
– β-barrel:alternating βαβ-units
4. Tertiary Structure
a) Refers to the 3D arrangement of a polypeptide chain forming combination of motifs/2⁰
shapes
b) It is not always possible to draw a clear distinction between 2° and 3° structure
c) Polypeptide chain conformation are stabilized by :
• disulphide bonds
• ionic bonds
• H- bonds
• hydrophobic interactions

5. Quaternary Structure
a) Protein consist more than one polypeptide chain. Each chain is called one subunit
b) Held together by similar covalent and non-covalent bonds as 3° structure

6. The Noncovalent Interactions That Dictate and Stabilize Protein Structures

a) van der Waals: 0.4 - 4 kJ/mol
b) hydrogen bonds: 12-30 kJ/mol
c) ionic bonds: 20 kJ/mol
d) hydrophobic interactions: <40 kJ/mol

7. The importance of correct protein folding

a) Protein folding is critical to the proper function of a protein
b) Incorrect folding (unfolded or misfolded) cause the protein to interact with other unwanted
protein, be non-fucntional, protease-sensitive,and forming aggregates
c) Aggregates protein allow its hydrophobic region to be exposed and interact with other
molecules when it is supposed to be buried inside
 d) Several neurodegenerative disorder caused by accumulation of such protein and aggregates
are:
 Alzheimer’s, Parkinsons , Huntington disease and more
e) Primary structure conveys all information necessary to produce the correct 3° structure
f) Nonetheless, in the protein-dense environment of a cell, proteins may begin to fold
incorrectly or may associate with other proteins before folding is completed
g) Special proteins called “chaperonins” aid in the correct and timely folding of many proteins
h) hsp70chaperoninsproduced in E.coli were the first protein chaperone discovered
i) TRiC is chaperonin produced in eukaryortes
j) Chaperonins exist in organisms from prokaryotes to humans

Fibrous and Globular Protein

1. Globular proteins: proteins which are folded to a more or less spherical shape
– they tend to be soluble in water and salt solutions
– most of their polar side chains are on the outside and interact with the aqueous
environment by hydrogen bonding and ion-dipole interactions
– most of their nonpolar side chains are buried inside
– nearly all have substantial sections of a-helix and b-sheet
– Examples
• myoglobin
• hemoglobin
2. Fibrous proteins: contain polypeptide chains organized approximately parallel along a single axis.
– consist of long fibers or sheets in shape
– tend to be mechanically strong
– are insoluble in water and dilute salt solutions
– Examples are
• keratin of hair and wool
• collagen of connective tissue of animals including cartilage, bones, teeth, skin,
and blood vessels
Protein Denaturation

1. Denaturation- Disruption of the protein structure that causes loss of biological activity
2. How?
 a) Heat
 ↑heat , ↑vibrations molecules, thus disrupt the bond
b) pH
 High or extreme low pH → alter charges on the protein side chain → cause to
reduces electrostatic interactions
c) Detergents
 Disrupt hydrophobic and electrostatic interactions
 Ex: urea, guadinine hydrochloride, sodium dodecyl sulfate (SDS),
mercaptoethanol

Protein Structure Determination

a) X-ray crystallography
 Used to determine 3° structure
 Very high purity protein can form crystals
 No crystal formation = no structure can be observe
 When beam of x-rays exposed on pure protein crystals, it creates a diffraction pattern
 The pattern is produced when the electrons in each atom scatter the x-rays.
 The information is extracted from the diffraction patterns through a mathematical
analysis known as Fourier series.

Protein Purification

1. Protein purification begins from homogenization of the sample, to centrifuge and separating the
protein of interest using chromatography or electrophoresis.
2. Homegenization is the process of releasing soluble protein fromcells and subcellular organelle.
There are various homogenization methods :
Method Description
Cause all cells to rupture
Cause all cells to rupture
Use sound waves to break open cells, but
leaves many organelles still intact
To release protein of interest that attached to
membrane
3. Differential centrifugation separates cell components on the basis of size and density using
different speed of centrifugation
 The larger and denser components will settled as sediment to form a pellet at the bottom
of the tube, while smaller, less dense components remain in suspension above, a portion
called supernatant.
 Protein of interest will be available in the supernantant,

4. Column chromatography separates different compounds based on interaction with stationary

phase and mobile phase. Three most common chromatography for protein separation used are:
 Size exclusion (Also called gel filtration chromatography)
 Separates molecules base on size

 Use cross linked gel particles (can also be porous solids) as st. phase
i. Carbohydrate polymer – dextran (Sephadex) or agarose (Sepharose)
ii. Polyacrilamide (BioGel)
  Smaller molecules are able to enter the pores , larger molecule elutes first,
followed by smaller ones
 Advantages: can be used to estimate MW by comparing with set of standards

 Affinity Chromatography
 Use specific binding ligand polymer/resin as a st phase
 The resin has a ligand where it would bind to the desired protein
 Protein that does not bind with the ligand will be eluted out with buffer
 The ligand-bound protein are eluted out using high concentration of ligand
solution
 Protein of interest will bind with ligand in mobile phase
 Produces very pure protein

 Protein-ligand interaction may be disrupted with a change in pH or ionic strength

 Ion exchange chromatography

 Similar to affinity chromatography but based on net charge (Cations or anion can
be separated)
 Molecules with specific charge are selectively bound to ligand on resin in the
column
 Ligand on resin has either +ve or –ve charge
a) -ve charged resin - cation exchanger
 The resin in the column are usually bound to Na+ or K+ ions
b) +ve harged resion - anion exchanger
 The resin in the column are usually bound to CI- ions
 Process:
a) The column will usually be equilibrated with buffer to filled the
exchange resin in the column with counter ions
b) A mixture of protein loaded on the column and flows through
c) Proteins that has opposite net charge will retain in the column by
exchanging places with the bound-counter ions
d) Non binding protein are eluted out
 e) The retained protein will then be eluted out using buffer that has pH that
removes the charge of the bound-protein or to one with higher salt
concentration (Desalting process)
 Example: Cation exchange chromatography , Anion exchange chromatography

5. List the differences of the following:

AFFINITY SIZE-EXCLUSION
CHROMATOGRAPHY CHROMATOGRAPHY

Separation by specific binding

Separation based on? of protein
Type of stationary Use cross linked gel particles and
phase used? porous solids as st. phase

Protein interest retained on Small molecules caught in pores,

Separation process: the column, others pass large molecules eluted first
through column followed by small molecules.

6. Electrophoresis: Separates molecules on gel medium by passing electrical current through the gel.
Proteins are separated on the gel based on their size, shape and charge
 Type of gel medium used:
– Agarose gel (for nucleic acids and small protein sample )
– Polyacrylamide gel (protein sample)
 Electrophoresis process:
a. Sample is applied to wells on the gel (agar)
b. An electric current is passed through the gel at a controlled voltage to achieve
desired separation
c. Protein with low MW will travel faster than protein with high MW
d. The separated protein on the gel is observed using UV-light box.

 Can be used to estimate the MW of the protein

by comparing sample with standards
 Based on studies , log MW is linearly related
to the mobility on SDS-PAGE

7. Specific uses of gel called Sodium dodecyl sulfate (SDS) Polyacrylamide Gel Electrophoresis
(PAGE).
 SDS-PAGE will denature sample protein at 3° and 4°structure into polypeptide chain at
random coil state. This means multisubunit protein can be analyzed as single unit type of
polypeptide chain
8. Electrophoresis: Isoelectric focusing (IEF)
 Makes use the principle of pI
 Isoelectric pH (pI) is the pH at which protein has no net charges
 At pI,+ve charge = -ve charge
  Different protein has different isoelectric point
 a gel is prepared with a pH gradient that parallels to the electric field
 Strong acid at anode
 Strong base at cathode
 PROCESS ;
a. Sample can be applied anywhere over the gel
b. Protein migrated through gel using electrical current. High voltage applied
2500V
c. Protein will encounter different region of pH as it migrates, charges on the
protein changes till it has no net charges
d. Once it reaches isoelectric point, protein no longer migrates
e. Each protein remains at the position on the gel corresponding to its pI

9. 2D- Electrophoresis :
Combine both isoelectric focusing and SDS PAGE

10. Determine the primary structure of a protein

STEP 1: Determining amino acid present and the what is proportion
 Break the amino acid by heating the solution in acid
a. 6M HCI at 100°C to 110° for 12-36 hours
b. Peptide bond will hydrolyze
 Use amino acid analyzer
a. HPLC, Ion exchange column
 b. Determine qualitative and quantity of amino acid
c. Identity of amino acid

STEP 2:Determine N-terminal to know how many polypeptide chains USING

 Sanger degradation method

a. Treat protein with thiol – breaks

disulfide bond in protein
b. Add 2,4-dinitro-fluorobenzene –
binds to the first amino acid of the
polypeptide
c. Identify the amino acid with 2,4-
dinitro-fluorobenzene as the first
amino acid

 using Edman Degradation

• Use Edman reagent : phenyl

isothiocyanate (PITC) reacts with N-
terminal residue
• The reagent will cleaves only one
amino acid without disrupting peptide
bond in the residues
• Further treatment with acid yields
thiazolinone derivative and can be
identified through HPLC or mass
spectrometry etc methods
•

STEP 3: (Degradation) and STEP 4 (Sequencing)

• break polypeptide chain into fragments for protein sequencing
• From 100 residues to 20-50 residues
• Can be cleave using enzymes or chemical reagent
– Trypsin
• cleaves peptide bond at amino acids that have +ve charge R-group
(lysine and arginine)
– Chymotrypsin
• cleaves at the aromatic amino acids (tyrosine , tryptophan and
phenylalanine)
– Cyanogen bromide
• cleave at sulfur bridge in methionine. The cleaved product is analyze by
HPLC chromatography and later proceed to complete sequencing.