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PTT 103 BIOCHEMISTRY
PROTEIN
Pn Khadijah Hanim Abdul Rahman
Protein structure
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Protein (polypeptides) are organic compound made of
amino acids arranged in a linear form and folded into
specific conformation
Protein - essential part of organisms
- most diverse functions
Function of proteins:
Catalysis (enzymes), structure (collagen, elastin),
movement (actin, tubulin), Defense (keratin,
fibrinogen, thrombin), regulation (insulin), transport
(function as carriers across membrane), storage
(casein), stress response
Protein structure
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When cell synthesizes a polypeptides, the chain
folded spontaneously
This folding is reinforced by variety of bonds
between the chain
In a complex structure of protein, several levels
of the structural organization of proteins :
a) primary structure
c) tertiary structure
b) secondary structure
d) quaternary structure
a) Primary structure
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Primary structure of protein is its unique sequence of
amino acids forming its polypeptide chain
Every polypeptides has a specific amino acid
sequence
the primary structure of a protein is starting from the
amino-terminal (N) end to the carboxyl-terminal (C)
end.
The interactions between amino acid residues
determine the protein’s 3-D structure and its
functional role.
Primary structure of enzyme lysozyme
b) Secondary structure
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Most proteins have segments of their polypeptide
chain repeatedly coiled or folded in patterns.
These coiled & folded referred as secondary
structure.
2 types of secondary structure :
- α-helix
stabilized by hydrogen bond
- β-pleated sheet between carbonyl & amino groups
in the polypeptide’s backbone
α-helix
 Rigid, rod like structure that forms when a
polypeptides chain twists into a right-handed
helical conformation
 Hydrogen bond form between amino group (NH) of each amino acid and the carbonyl group of
the amino acid four residue away (H bond form
between 4 amino acid)
 There are 3.6 a.a residues per-turn helix.
 R group extend outward from the helix
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Because of several structural constraints,
certain a.a do not foster α-helical formation
Glycine R group is too small that the p.p chain
may be too flexible.
Proline: contains a rigid ring that prevents NCα bond from rotating. Proline also has no NH group available to form H bonds that are
crucial in α-helix
Bulky R groups a.a are also incompatible with
α-helix structure.
β-pleated sheet
 Form when two or more polypeptide chain
segments line up side by side
 Each individual segment = β-strand
 Each β-strand is fully extended
 β-pleated sheet stabilized by hydrogen bonds
form between the polypeptide backbone N-H and
carbonyl groups of adjacent chains
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Two β-pleated sheet :
- parallel - polypeptide chain arranged in same
direction
- antiparallel - polypeptide chain arranged in
opposite direction
- more stable because fully
colinear H bonds form.
Usually mixed parellal-antiparallel β-pleated
sheet observed in proteins
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combination of α-helix and β-pleated sheet
secondary structure = supersecondary structure
Supersecondary structure :
a) βαβ unit two parallel β-pleated sheets
connected by α-helix
fragment
b) β-meander two antiparallel β-sheets are
connected by polar amino acids and glycines
to effect an abrupt change in direction of the
polypeptide chain (reverse or β-turns)
b meander
c) αα-units two α-helices separated by loop
or nonhelical segment
d) β-barrel- form when
various β-sheet
configurations fold back on
themselves
e) Greek key- antiparallel βsheet doubles back on itself
in a pattern that resemble a
common greek pottery
design
c) Tertiary structure
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The term tertiary structure refers to the unique
3-D conformations that globular protein
assume as they fold into their native structures
(biologically active).
The α-helices and β-pleated sheets are folded
into compact globule.
Protein folding occurs as consequence of
interactions between the side chains in their
primary structure
Tertiary structure has several
important features:
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Many p.p fold in such fashion that a.a residues that
are distant from each other in the primary structure
come into close proximity
Because of efficient packing as the p.p chain folds,
globular protein are compact. Most water molecules
are excluded from protein’s interior making
interactions between both polar and non-polar groups
possible.
Large globular protein contain several compact units
called domains. Domains: structural independent
segments that have specific functions.
Interactions that stabilize tertiary
structure
1) Hydrophobic interactions
 As polypeptide folds, amino acids with
hydrophobic (nonpolar) side chain are brought
close to each other, out of contact with water.
2) Electrostatic interactions
 Interaction occurs between ionic groups of
opposite charge (referred as salt bridge)
3) Hydrogen bonds
 Large number of hydrogen bond form within a
protein’s interior and on its surface
 Examples of amino acid side chains that may
hydrogen bond to each other:
 Two alcohols: ser, thr, and tyr.
Alcohol and an acid: asp and tyr
Two acids: asp and glu
Alcohol and amine: ser and lys
Alcohol and amide: ser and asn
4) Covalent bond
 Created by chemical reactions that alter a
polypeptide's structure during or after its
synthesis
 eg. Disulphide bond (strong linkage)
 Protect protein structure from adverse changes
in pH or salt concentrations
d) Quaternary structure
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Proteins consist of two or more polypeptide chains
aggregated into one functional macromolecules
Many proteins, esp those with high molecular weight
are composed of several polypeptide chains.
In proteins that consist of more than 1 polypeptide
chain, each polypeptide is called subunit
Polypeptide subunits assemble and held together by
noncovalent interaction eg H bonding, hydrophobic
effect, electrostatic interaction
Loss of protein structure
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Considering the small differences in the free
energy of folded and unfolded protein- protein
structure is sensitive to environment factors.
Many physical & chemical agents can disrupt
protein’s native conformation
The process of structure disruption =
denaturation
Denaturing conditions :
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Strong acid or base – changes in pH result in
protonation of some protein side group, which
alter/disrupt hydrogen bonding & salt bridge. As a
protein approaches its isoelectric point, it becomes
insoluble and precipitates from solution.
Organic solvents – water-soluble organic solvents eg.
Ethanol interfere with hydrophobic interaction
because they interact with nonpolar R groups and
form H bond with water and polar protein group.
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Detergents – these amphiphatic molecules
disrupt hydrophobic interaction causing
proteins to unfold into extended polypeptide
chains
(amphiphatic = contain nonpolar and polar
components)
Reducing agents – eg. Urea, βmercaptoethanol, will convert disulfide bridge
(S-S) to sulfhydryl group (SH)
urea disrupt H bond & hydrophobic interaction
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Heavy metal ions – mercury (Hg+) and lead
(Pb2+) disrupt salt bridge by forming ionic bond
with negatively charge group.
Temperature change – as temp increase, the rate
of molecular vibration increase. So weak H
bond will be disrupt and protein will unfold.
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Mechanical stress – stirring & grinding actions
disrupt the delicate balance of forces that
maintain protein strcuture.
eg. Foam formed when egg white is beaten
vigorously contains denatured protein
Fibrous proteins
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Fibrous protein exist as a long stranded
molecules
Contain high proportions of secondary
structures ; α-helices and β-pleated sheets
Most are structural protein
eg. α-keratin, collagen, silk fibroin
α-keratin
 Found in hair, wool, skin, horns, fingernails is
an α-helical polypeptides
 Each polypeptide has three domain :
- an amino terminal ‘head’
- a central rodlike α-helical domain
- a carboxyl terminal ‘tail’
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Two keratin polypeptides associate to form =
coiled coil dimer
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Two antiparallel rows of these dimer form a
supercoiled structure called a protofilament (H
bonds & disulfide bond aid the formation of
protofilament)
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Hundreds of filaments, each containing 4
protofilaments form macrofibril
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Each hair cells (fiber) contain several
macrofibrils
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Physical properties of the α-keratins are reflected in
their amino acids compositions.
They have a regular α-helix structure because they
lack of proline and have alanine and leucine.
Because R groups are on the outside of the α-helices,
the high hydrophobic a.a content makes it insoluble in
water.
Its cys residues and the formation of interhelix
disulfide bridges make it resistant to stretching.
Collagen
 The most abundant protein in vertebrates
 Synthesized by
- connective tissue cells
 mostly found in fibrous tissues such as :
tendon, ligament and skin, in cornea, cartilage,
bone, blood vessels, the gut
 Extremely strong
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Collagen is composed of three lefthanded polypeptide helices that are
twisted around each other to form a
triple helix (stabilized by hydrogen
bonding)
The amino acid composition of collagen
is distinctive
- high content of glycine, proline and
lysine
- very little amount of cysteine (unlike αkeratin)
Silk fibroin
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Silk protein
- form spider webs, cocoon and nests
- consist of the fibrous protein fibroin
Considered to be β-keratin
- polypeptide chains arranged in antiparallel βpleated sheet comformation
Its primary structure mainly consists of the amino
acid sequence (Gly-Ser-Gly-Ala-Gly-Ala)n
Because the β-pleated sheets are loosely bonded to
each other, they slide over each other easily. This
arrangements gives silk fibers their flexibility.
Globular protein
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Globular protein have a spherical shape,
compact and water-soluble
In their function, usually require them to bind
precisely to other molecules
Each protein has a unique and complex surface
that contains cavities and clefts whose
structure is complementary to specific ligands.
After ligand binding, a conformational change
occurs in the protein that is linked to a
biochemical event.
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Most enzymes are globular
Myoglobin & hemoglobin are typical example
of globular protein
Both are hemoprotein and each is involved in
oxygen metabolism
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Unlike fibrous proteins which only play a structural
function, globular proteins can act as:
1) Enzymes, by catalyzing organic reactions taking
place in the organism in mild conditions and with a
great specificity.
2) Messengers, by transmitting messages to regulate
biological processes. This function is done by
hormones, i.e. insulin etc.
3) Transporters of other molecules through membranes
4) Stocks of amino acids.
5) Regulatory roles are also performed by globular
proteins rather than fibrous proteins.
a) Myoglobin
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Myoglobin - oxygen-transport protein found in
the muscle tissue of vertebrates in general and
in almost all mammals.
Diving mammals – high conc of myoglobin in
the muscle tissue (brown in colour)
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has the ability to store oxygen by binding it to
an iron atom (heme)
It is found abundantly in the tissues of diving
mammals, e.g., the whale, the seal, and the
dolphin.
High concentrations of myoglobin in these
animals, allows them to store sufficient oxygen
to remain underwater for long periods.
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Protein component = globin (single
polypeptides chain (153 a.a) with 8 section of
α-helix)
Each myoglobin molecule contains one heme
prosthetic group
Each heme consist of porphyrin ring with Fe2+
in the center
Free heme [Fe2+] has a high affinity for O2 and
is irreversibly oxidized to form hematin [Fe3+]
b) Hemoglobin
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Hemoglobin is a roughly spherical molecule
found in red blood cells
Function = transport oxygen from lung to
every tissues in the body
Composed - two α-chain
- two β-chain
The protein contain four subunits, designated α
and β. Each subunit contain a heme group that
bind with oxygen
Protein technology: Purification of
protein
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After protein has been isolated, several
methods can be used to enhance purification.
Salting out
Technique in which high concentrations of
salts such as [(NH4)2SO4] are used to
precipitate proteins.
This technique removes impurities
Dialysis is routinely used to remove lowmolecular weight impurities such as salts,
solvents and detergents.
Chromatography
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Chromatography techniques can be used to
separate protein mixtures on the basis of
molecular properties, such as size, shape and
weight or binding affinities.
In all chromatographic methods, protein
mixture is dissolved in liquid (mobile phase).
As protein molecules passed thru stationary
phase (solid matrix), they separate because of
different distributions between 2 phases.
Gel-filtration chromatography
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A column packed with gelatenous polymer separates
molecules according to size and shape.
Molecules larger than gel pores are excluded and
move faster thru the column.
Molecules that are smaller than gel pores diffuse in
and out of pores- their movement thru the column is
retarded.
The smaller the molecular weight, the slower they
move.
Differences in these rates separate protein mixture
into bands, which are then collected separately.
Ion-exchange chromatography
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Separates protein based on their charge.
Anion-exchange resins- consist of +vely
charged materials. Binds reversibly with
protein’s negatively charged groups.
Cation-exchange resins: bind positively
charged groups.
After proteins that do not bind to resin are
removed, the protein of interest is recovered by
appropriate change in solvent pH/salt conc. (a
change in pH alters the charge).
Affinity Chromatography
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Uses unique properties of proteins.
Uses a special non-covalent binding affinity
between protein and special molecule- ligand.
Ligand is covalently bound to insoluble matrix
which is placed in the column.
After nonbinding protein molecules have
passed thru the column, the protein of interest
is removed by altering the conditions that
affect binding (pH/salt conc.).
Electrophoresis
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Proteins are electrically charged- they move in an electric field.
In electrophoresis- molecules separate from each other because of
differences in their net charge.
Positive net charge migrate toward the –vely charged electrode and
negative net charge migrate to +vely charged electrode. No charge=not
moving.
Electrophoresis using polyacrylamide/agarose gel.
The gel separate proteins on the basis of their molecular weight and shape.
During purification, specific bands may be excised from the gel after
visualization. Each protein containing fragment is then eluted with buffer.
Gel electrophoresis also used to assess the purity of protein samples.
Staining the gel with dye Coomassie blue is commanly used to assess the
success of a purfication step.