PROTEINS

BIOMEDICAL IMPORTANCE
Proteins are physically and functionally complex
macromolecules that perform multiple critically
important roles.
Cytoskeleton

Actin and
myosin filaments
Hemoglobin
Antibodies
Enzymes
hormones

PURIFICATION OF PROTEINS AND

PEPTIDES
= it must be purified prior to analysis
Highly purified protein is essential for the detailed
examination of its physical and functional properties
Classic approaches exploit differences in relative
solubility of individual proteins as a
function of pH (isoelectric precipitation), polarity
(precipitation with ethanol or acetone), or salt
concentration
(salting out with ammonium sulfate).

Column Chromatography
employs as the stationary phase small spherical beads
of modified cellulose,
acrylamide, or silica whose surface typically has been
coated with chemical functional groups

COLUMN CHROMATOGRAPHY

 Partition Chromatography
Size Exclusion Chromatography
Ion Exchange Chromatography
Hydrophobic Interaction Chromatography
Affinity Chromatography
Absorption Chromatography
Ion Exchange Chromatography
Hydrophobic Interaction Chromatography
Affinity Chromatography

Protein Purity Is Assessed by Polyacrylamide

Gel Electrophoresis (PAGE)
The most widely used method for determining the purity
of a protein is SDS-PAGEpolyacrylamide gel
electrophoresis (PAGE) in the presence of the anionic
detergent sodium dodecyl sulfate (SDS).

WHO IS FREDERICK SANGER?

SANGER WAS THE FIRST TO DETERMINE THE
SEQUENCE OF A
POLYPEPTIDE

 Pehr Edman introduced phenylisothiocyanate (Edman's

reagent) to selectively label the amino-terminal residue
of a peptide

Traditional Architecture

Wood, brick, nails, glass

Temperature, earthquakes

How many people?

How many doors and windows?

Spanish, Victorian,
1950's blocky science building
Julia Morgan

Form
fits
function

Materials
Environmental Factors
Population Factors

Molecular Architecture

Amino acids, cofactors

Temperature, solubility

# partner proteins, # reactants

Portals

Passages for substrates and reactants

Motifs/Styles

Conserved domains or protein folds

Architect

Evolution

Levels of Protein Structure

PRIMARY STRUCTURE
sequence of amino acid
understanding the primary structure of protein is
important

PEPTIDE BOND
- Amino acids are joined covalently, which are
amide linkages between the - carboxyl group of
one amino acid and the -amino group of another

SECONDARY STRUCTURE
Does not assume a random three-dimensional structure
Forms regular arrangements of amio acids
Alpha helix
Beta sheet
Beta bend/turn

Alpha () helix
Most commonly found in nature
spiral structure consisting of tightly packed, coiled
polypeptide backbone core
With side chains of the component amino acids
extending outward from the central axis to avoid
interfering sterically with each other
Example: keratin (fibrous protein); myoglobin

-helix
Stabilized by extensive hydrogen bonding between the
peptide bond carbonyl oxygens and amide hydrogens
Each turn of an alpha helix contains 3.6 amino acids
Proline disrupts an alpha helix
( glutamate,
apartate, histidine, lysine or arginine)
Trytophan, valine, isoleucine ( bulky side chains)

Beta sheet
All peptide bond components are involved in hydrogen
bonding
The surfaces of beta sheets appear pleated called pleated sheets

Comparison of a - sheets and - helix

Unlike - helix - sheets are composed of two or more
peptide chains (- strands), or segment of polypeptide
chains, which are almost fully extended.
In - sheets the hydrogen bonds are perpendicular to
the polypeptide backbone.

Parallel and antiparallel sheets

A - sheets can be formed from 2 or more polypeptide
chains or segments of other or parallel to each other
When the hydrogen bonds are formed between the
polypeptide backbone of separate polypeptide chain
they are termed interchain bonds.
A - sheets can also be formed by a single polypeptide
chain folding back on itself
- sheets in globular protein always have right handed
curl or twist when viewed along polypeptide backbone

- Bends ( reverse turns,- turns)

- bends reverse the direction of a polypeptide chain,
helping it from a compact globular shape
Generally composed of 4 amino acids which may be
proline- the amino acid that causes a kink in the
polypeptide chain
Glycine the amino acid with the smallest R-group is also
frquently found in - bends
- bends are stabilized by the formation of hydrogen
ionic bonds

Non repetitive secondary structure

Approximately one half of an average globular protein is
organized into repetitive structure such as -sheets and
- helix
Not random but rather simpply has les regular structure
than those describe above

Supersecondary structures (motif)

These form primarily the core region that is interior of
the molecule
Connected by loop regions at the surface of the protein
Usually produced by packing side chains from adjacent
secondary structural elements close to each other

Tertiary structure of Globular Proteins

Tertiary refers both to the folding of the domains and to
the final arrangements of domain in the polypeptide
Globular chains in aqueous solution is compact with
high density of atoms in the core
Hydrophobic side chains is buried in the interior, where as hydrophilic groups are
generally found on the surface of the molecule

Domains
Fundamental functional and three dimensional structural
units of polypeptides
Polypeptide chains >200 amino acids has 2 domains
Each domain has the characteristic of a small
structurally independent of the other domains in
polypeptide chains

4 types of interactions cooperate in

stabilizing the tertiary structure proteins
Disulfide bonds
Covalent linkage formed from sulfhydryl groups of each 2
cystine residue
Hydrophobic interactions
amino acid with non polar side chains tend to be located in the
anterior of the polypeptide molecule where they associate with other
hydrophobic amino acids

 Hydrogen bonds
Amino acid side chains containing oxygen or nitrogen- bound
hydrogen such as in the alcohol groups of serine and
threonine, can form hydrogen bonds with electron rich atoms
such as oxygen of a carboxyl or carbonyl group of a peptide
bond
Ionic interactions
Negatively charged groups such as the carboxylate in the ide chains
of aspartate or glutamate can interact with the positively charged
groups such as the amino group in the side chain of lysine

Protein folding
Occurs within the cell in second or minute, employs a
shortcut through the maze of a folding possibilities
As peptides folds its amino acid side chains are attracted
and repulsed according to their chemical properties

Denaturation of proteins
Protein denaturation results in the unfolding and
disorganization of the protein secondary and tertiary
structures which is not accompanied by hydrolysis of
peptide bonds.
Denaturing agent include heat, organic solvents,
mechanical mixing, strong acids or bases, detergents
and ions of heavy metals.

Role of chaperones in protein folding

The chaperones also known as heat shock proteinsinteract with the polypeptide at various stages during
the folding process
Some chaperones are important in keeping the protein
unfolded until its synthesis I finished, or act as catalyst
by increasing the rates of the final stages in the folding
process
Others protect proteins

QUATERNARY STRUCTURE OF PROTEINS

consist of 2 or more polypeptide chains that may be
structurally identical or totally unrelated
Subunits are held together by non covalent interaction
Subunits may function independently or cooperatively

PROTEIN MISFOLDING
Is a complex , trial and error process that can sometimes
result in improperly folded molecules.
It is usually tagged and degraded with the cell

Amyloid disease
Accumulation of insoluble, spontaneously aggregating
proteins called amyloids, has been implicated in many
degenerative disease particularly in the age related
neurodegenerative disorder, Alzheimer disease
Dominant component of the amyloid plaque that
accumulate in Alzheimer's disease is amyloid (A) a
peptide containing 40-42 amino acids residues
A second biologic factor involved the development of
Alzheimer disease is the accumulation of the
neurofibrillary tangles inside neurons

Prion Disease
The prion protein (PrP) has been strongly implicated as
the causative agent of transmissible spongiform
encephalopathies (TSEs) including Creutzfeldt- Jakob
disease in humans, scarpie in sheep, bovine spongiform
encephalopathy in cattle mad cow disease
It is highly resistant in proteolytic degradation and
tends to form insoluble aggregates of fibrils similar to
the amyloid found in some other diseases of the brain

 PrPSC is the infectious protein

PrPc is the noninfectious form
it has been observed that a number of-helices present in non
infectious PrPc are replaced by - sheets in the

infectious form
The infective agent is thus an altered version of a
normal protein, which acts as a template for
converting the normal protein to the pathogenic
conformation
The TSEs are invariably fatal, and no treatment is
currently available that can alter this outcome.