BIO462 664336629.

docx Aisya Noordin

1.1 PROTEIN STRUCTURE

LEVEL OF PROTEIN STRUCTURE

 Primary structure  the linear sequence of amino acids
joined together by peptide bonds
 Secondary structure  the regular folding of regions of the
polypeptide chains (α-helix, β-pleated)
 The tertiary structure  describes how the entire protein
molecule coils into an overall three-dimension shape
 The quaternary structure  describes how different protein
molecule come together to yield large aggregate structures.

PRIMARY STRUCTURE OF PROTEINS

 The primary structure refers to the sequence of the different amino acids of the peptide or protein that held together
by peptide bonds.
 The two ends of the polypeptide chain are referred to as the carboxyl terminus (C-terminus) and the amino terminus
(N-terminus)

SECONDARY STRUCTURE
 Secondary structures are defined by patterns of hydrogen bonds between the carboxyl groups and the amino groups
of different amino acids in the polypeptide backbone.
 These hydrogen bonds can twist and stabilize the amino chain so that it is woven together into:
 A series of sheet  β-pleated sheet
 A series of spiral  α-helix
 A single chain of amino acids can be woven into pleated sheets in one area, and alpha helixes in a different area.

α-helix β-pleated sheet

 This structure resembles a coiled spring and is  Composed of two or more
secured by hydrogen straight chains that are hydrogen
bonding in the bonded side by side.
polypeptide chain.  If the amino terminus is on the
 The helix is stabilized by same end of each chain, the
hydrogen bonds sheet is termed parallel, and if
between the N-terminal the chains run in the opposite
of one amino acid and direction, the sheet is termed
the C=O group on the antiparallel.
4th amino acid away
from it.

1|Page
 BIO462 664336629.docx Aisya Noordin

TERTIARY STRUCTURE QUATERNARY STRUCTURE

 Determined by a variety of bonding interaction  Clustering of several individual peptide or protein chains
between the side chain on the amino acids. into a final specific shape
 Bonding interaction  A variety of bonding interaction including hydrogen
may be stronger bonding, salt bridge, and disulphide bonds hold the
than the hydrogen various chains into a particular geometry
bonds between the  There are two categories of proteins with quaternary
amide group holding structure
the helical structure.  Fibrous
 As a result, bonding  Globular
interaction between
side chain may
cause a number of
folds, bends, and
loops in the protein
chain

GLOBULAR PROTEIN FIBROUS PROTEINS

 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.
 Examples:
- myoglobin
- hemoglobin
 Fibrous proteins: contain polypeptide chains organized
approximately parallel along a single axis. They :
- consist of long fibers or large sheets
- tend to be mechanically strong
- play important structural roles in nature
 Examples:
- keratin of hair and wool
- collagen of connective tissue of animals including
cartilage, bones, teeth, skin, and blood vessels

DENATURATION OF PROTEIN
 The tertiary structure of protein is result of many intra-molecular interactions that can be disrupted by change of the
environment, causing the protein to become denatured.
 Enzymes lose all catalytic activity when denatured
 Denaturation can be brought about by heat, pH, detergent and urea

2|Page
 BIO462 664336629.docx Aisya Noordin

1.2 DETERMINATION PROTEIN STRUCTURE

PROTEIN FOLDING DOMAINS

 Protein must fold to function.  Domains have been described as:
 Some diseases are caused by misfolding. - Geometrically separate regions of protein structure
 Eg: mad cow disease - Structure with a recognized function
 caused by the misfolding of a protein known as - Sub-sequences that can fold independently into a
PrP  stable structure
 affects cow’s nervous system – lose control.

PROTEIN STRUCTURE DETERMINATION

 High resolution structure determination
- X-ray crystallography ~1A
- Nuclear magnetic resonance (NMR) ~ 1-2.5A
 Low resolution structure determination
- Cryo-EM (electron microscopy) ~10-15A

3|Page
 BIO462 664336629.docx Aisya Noordin

X-RAY CRYSTALLOGRAPHY X-RAY DIFFRACTION

 More accurate 1. Crystallize protein of interest
 Extremely pure protein sample is needed 2. Expose crystallized protein
 Protein sample must form crystal to X-ray source
 Many protein are not amenable to crystallization at (wavelength 1.5
all angstroms)
3. Record diffraction pattern
NUCLEAR MAGNETIC RESONANCE (NMR) 4. Use intensities and
 Used in advanced medical imaging techniques, such positions of spots to
as in magnetic resonance imaging. determine atom identity
 Fairly accurate and position.
 No need for crystal
 Limited to small and soluble protein only

4|Page
 BIO462 664336629.docx Aisya Noordin

Determining the Primary Structure of a Protein

 Step 1: to establish which amino acids are present and in what proportions  amino acid analyzer.
 Step 2: identities of the N-terminal and C-terminal are determined.
 Step 3 and 4: protein is cleaved into smaller fragments and the amino acid sequence is determined  Edman
degradation.

Edman Degradation

Edman degradation, developed by

Pehr Edman  method
of sequencing amino acids in a
peptide.
The amino-terminal residue is
labeled and cleaved from the
peptide without disrupting the
peptide bonds between other
amino acid residues.

PROTEIN SIZE
 In general, proteins contain > 40 residues
- Minimum needed to fold into tertiary structure
 Usually 100-1000 residues
 Percent of each AA varies
 Proteins separated based on differences in size and composition
 Proteins must be pure to analyze, determine structure/function

3.2 PROTEIN PURIFICATION

5 |Protein
P a g e purification is a series of processes intended to isolate one or a few proteins from a complex
mixture, usually cells, tissues or whole organisms.
 BIO462 664336629.docx Aisya Noordin

 Many different proteins exist in a single cell.

 A detailed study of the properties of any one protein requires a homogeneous sample consisting of only one kind
of molecule.
 Separation techniques focus on size, charge, and polarity – the source differences between molecules.

Overview of protein analysis

 Homogenization.
 Differential centrifugation.
Analysis

Sample preparation
HOMOGENIZATION
 Before the real purification steps can begin, the protein
must be released from the cells and subcellular organelles.
 Homogenizer = a thick-walled test tube and tight-fitting
plunger.
 After the cells are homogenized, they are subjected to
differential centrifugation.
 Cell are lysed and ruptured

Differential centrifugation

SEPARATING PROTEINS: CHROMATOGRAPHY TECHNIQUE

CHROMATOGRAPHY

6|Page
 BIO462 664336629.docx Aisya Noordin

 The expansion of the protein band in the mobile phase is caused by separating of protein with different properties
and diffusion spreading
 As the length of column increase, the resolution of two type of protein improves
1. Ion exchange chromatography
• Anion
• Cation
2. Gel filtration chromatography (size exclusion)
• Separates based on size
3. Affinity chromatography
• Affinity of ligands
ION EXCHANGE CHROMATOGRAPHY
o Separate based on net charge of proteins
o Stationary phase = chemically modified to
include charged groups
o Affected by pH
o Anion exchangers
- positively charge resin
o Cation exchangers
- negatively charge resin

GEL FILTRATION CHROMATOGRAPHY (size exclusion)

o Size/molecular exclusion
chromatography
o Stationary phase = column
matrix is a cross-linked
polymer /gels with pores
of particular size
o Molecules separate based
on size
 Small molecules caught in
pores
 Large molecules pass
through

AFFINITY CHROMATOGRAPHY

7|Page
 BIO462 664336629.docx Aisya Noordin

 Separate protein by their binding specificities

 Stationary phase = column matrix is covalently linked to some compound called ligand
 Protein retained on the column are those that bind specifically to a ligand, while other proteins pass through column

ELECTROPHORESIS
 Separation of protein based on migration of ions in an electric field
 The positively charged proteins move towards the negative electrode (cathode) – attract cation.
 The negatively charged proteins move toward the positive electrode (anode) – attract anion.
 A protein at its pI (neutral) will not migrate in either direction

SDS-PAGE ISOELECTRIC FOCUSING

 Sodium dodecyl  Another variation of gel electrophoresis
sulfate  Different proteins have different isoelectric points (pH at
polyacrylamide gel which a particular molecule carries no net electrical
electrophoresis charge)
(SDS-PAGE), is a - At the pI, the number of +ve charges exactly
technique widely balances the number of –ve charges.
used in
 In isoelectric focusing, a gel is prepared with a pH
biochemistry,
gradient that parallels the electric field gradient.
forensics, genetics
and molecular
biology.
 To separate
proteins according
to their electrophoretic mobility (a function of length
of polypeptide chain or molecular weight)
 Following electrophoresis, the gel may be stained
(most commonly with Coomassie Brilliant Blue R-250
or silver stain), allowing visualization (UV lamp) of
the separated proteins
 It is common to run molecular weight size markers of
known molecular weight in a separate lane in the gel,
in order to calibrate the gel and determine the weight
of unknown proteins by comparing the distance
traveled relative to the marker

3.3 ENZYME PURIFICATION

ENZYME PURIFICATION

8|Page
 BIO462 664336629.docx Aisya Noordin

 Extraction of a single enzyme from cell/ tissues may contain more than 1000 different proteins and lots of other
biomolecules
 Each may have steps that are specific to the properties of the enzyme
 There are features that tend to be common in methods of recovery of proteins – enzyme are polymers of amino
acids with many common features

WHY PURIFY ENZYME?

 To understand what is going on in a cell and between cell – metabolic and regulatory pathway
 To study reaction, kinetics of enzyme, etc.
 To understand deviations in normal metabolism or regulation process due to abnormal enzyme
 To use enzyme/protein for industrial purposes

REQUIREMENT OF ENZYME PURIFICATION LOCATION OF ENZYMES

 pH  Intracellular enzyme: cytoplasmic, organelle
 Temperature  Extracellular enzyme: released by the cell
 Salt concentration  Cell-wall bound: attach to a cell membrane
 Oxygen sensitivity
EXTRACTION TECHNIQUE
 Storage
 Extraction must be carried out as soon as possible
 Mechanical forces
after sample are obtained.
* Each enzyme requires a specific strategy for purification
 Otherwise, freeze / cool samples
 Cooling will reduce protease activity and general
tissues breakdown

PURIFICATION OF ENZYME ISOLATION OF ENZYMES

1. Precipitation methods  Centrifuge
2. Separation based on molecular size - Low speed (<3000rpm)
3. Separation based on charge - Moderate speed (<20,000 rpm)
4. Separation based on specific interaction with other - Very high speed (>20,000 rpm)
biomolecule  Filtration
- Cotton wool
- Membrane (>1µm)
SEPARATE CELL DEBRIS

PRECIPITATION / SALTING OUT

 Most common method for purification after extraction and isolation
 3 general methods
- Salting out of protein – ammonium precipitation
- Addition of organic solvent – ethanol, acetone
- Additional of polymers with high molecular weights – polyethylene glycol

9|Page
 BIO462 664336629.docx Aisya Noordin

SALTING OUT OF PROTEIN

 A method of separating protein based on the principle that protein are less soluble at high salt concentration
 The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein
√ There are hydrophobic amino acids and hydrophilic amino acids in protein molecules.
 After protein folding in the aqueous solution – hydrophilic amino acids interact with the molecules of H 2O and
allow protein to form hydrogen bond with surrounding water molecule.
 When the salt concentration is increased, some of the water molecules are attracted by the salt ion – decrease
the number of water molecule available to interact with the charged part of the protein.
 Protein molecules coagulates by forming hydrophobic interaction with each other
 Known as salting out

DIALYSIS
 Carried out after salting out to remove residual  used to desalt and concentrate protein sample
 Removed ammonium sulphate and other small molecular weight compounds such as sugar or organic acids
 Concept:
 Separate of big and small molecule through permeable membrane in buffer / distilled water

10 | P a g e