ERT106 BIOCHEMISTRYERT106 BIOCHEMISTRY

PROTEINPROTEIN

Pn Syazni Zainul KamalPn Syazni Zainul Kamal

Protein structureProtein structure

Protein (polypeptides) are organic compound Protein (polypeptides) are organic compound made of amino acids arranged in a linear form made of amino acids arranged in a linear form and folded into specific conformationand folded into specific conformation

Protein - essential part of organisms Protein - essential part of organisms

- participate virtually in every - participate virtually in every process process within cellswithin cells

Protein structureProtein structure When cell synthesizes a polypeptides, the chain When cell synthesizes a polypeptides, the chain

folded spontaneouslyfolded spontaneously This folding is reinforced by variety of bonds This folding is reinforced by variety of bonds

between the chainbetween the chain In a complex structure of protein, several levels In a complex structure of protein, several levels

of the structural organization of proteins :of the structural organization of proteins :

a) primary structurea) primary structure c) tertiary structurec) tertiary structure

b) secondary structureb) secondary structure d) quaternary structured) quaternary structure

a) Primary structurea) Primary structure

Primary structure of protein is its unique Primary structure of protein is its unique sequence of amino acids forming its sequence of amino acids forming its polypeptide chainpolypeptide chain

Every polypeptides has a specific amino acid Every polypeptides has a specific amino acid sequencesequence

the primary structure of a protein is starting the primary structure of a protein is starting from the amino-terminal (N) end to the from the amino-terminal (N) end to the carboxyl-terminal (C) end carboxyl-terminal (C) end

Primary structure of enzyme lysozyme

b) Secondary structureb) Secondary structure Most proteins have segments of their polypeptide Most proteins have segments of their polypeptide

chain repeatedly chain repeatedly coiledcoiled of of folded folded in patterns.in patterns. These coiled & folded referred as These coiled & folded referred as secondary secondary

structure.structure. 2 types of secondary structure : 2 types of secondary structure :

- - αα-helix-helix stabilized by hydrogen bondstabilized by hydrogen bond

- - ββ-pleated sheet-pleated sheet between carbonyl & amino groupsbetween carbonyl & amino groups

in the polypeptide’s in the polypeptide’s backbonebackbone

αα-helix -helix Rigid, rodlike structure when a polypeptides Rigid, rodlike structure when a polypeptides

chain twists into a right-handed helical chain twists into a right-handed helical conformationconformation

Hydrogen bond form between Hydrogen bond form between amino groupamino group of of each amino acid and the each amino acid and the carbonyl groupcarbonyl group of the of the amino acid four residue away (H bond form amino acid four residue away (H bond form between 4 amino acid)between 4 amino acid)

R group extend outward from the helixR group extend outward from the helix

ββ--pleated sheetpleated sheet Form when two or more polypeptide chain Form when two or more polypeptide chain

segments line up side by sidesegments line up side by side Each individual segment = Each individual segment = ββ--strandstrand Each Each ββ--strand is fully extendedstrand is fully extended ββ--pleated sheet stabilized by hydrogen bonds pleated sheet stabilized by hydrogen bonds

form between the polypeptide backbone N-H and form between the polypeptide backbone N-H and carbonyl groups of adjacent chainscarbonyl groups of adjacent chains

Two Two ββ--pleated sheet : pleated sheet :

- parallel- parallel - polypeptide chain arranged in same - polypeptide chain arranged in same direction direction

- antiparallel - antiparallel - polypeptide chain arranged in - polypeptide chain arranged in opposite direction opposite direction

- more stable - more stable Usually mixed parellal-antiparallel Usually mixed parellal-antiparallel ββ--pleated pleated

sheet observed in proteinssheet observed in proteins

combination of combination of αα-helix and -helix and ββ--pleated sheet pleated sheet secondary structure = secondary structure = supersecondary structuresupersecondary structure

Supersecondary structure :Supersecondary structure :

a) a) ββααββ unitunit two parallel two parallel ββ-pleated sheets -pleated sheets connected by connected by αα-helix -helix

fragmentfragment

b) b) ββ-meander-meander two antiparallel two antiparallel ββ-sheets are -sheets are connected by polar amino acids and glycines connected by polar amino acids and glycines to effect an abrupt change in direction of the to effect an abrupt change in direction of the polypeptide chain (reverse or polypeptide chain (reverse or ββ-turns)-turns)

meander

c) c) αααα-units-units two two αα-helices separated by loop -helices separated by loop or nonhelical segmentor nonhelical segment

d) d) ββ--barrel form when various barrel form when various ββ--sheet sheet configurations fold back on themselvesconfigurations fold back on themselves

e) Greek key antiparallel e) Greek key antiparallel ββ--sheet doubles back sheet doubles back on itself in a pattern that resemble a common on itself in a pattern that resemble a common greek pottery designgreek pottery design

c) Tertiary structurec) Tertiary structure

Three-dimensional structure of a single protein Three-dimensional structure of a single protein moleculesmolecules

The The αα-helices and -helices and ββ-pleated sheets are folded -pleated sheets are folded into compact globule.into compact globule.

Protein folding occurs as consequence of Protein folding occurs as consequence of interactions between the side chains in their interactions between the side chains in their primary structureprimary structure

Interactions that stabilize tertiary Interactions that stabilize tertiary structurestructure

1) 1) Hydrophobic interactionsHydrophobic interactions As polypeptide folds, amino acids with As polypeptide folds, amino acids with

hydrophobic (nonpolar) side chain are brought hydrophobic (nonpolar) side chain are brought close to each other, out of contact with water.close to each other, out of contact with water.

2) 2) Electrostatic interactionsElectrostatic interactions Interaction occurs between ionic groups of Interaction occurs between ionic groups of

opposite charge (referred as salt bridge)opposite charge (referred as salt bridge)

3) 3) Hydrogen bondsHydrogen bonds Large number of hydrogen bond form within a Large number of hydrogen bond form within a

protein’s interior and on its surfaceprotein’s interior and on its surface Examples of amino acid side chains that may Examples of amino acid side chains that may

hydrogen bond to each other:hydrogen bond to each other: Two alcohols: ser, thr, and tyr.Two alcohols: ser, thr, and tyr.

Alcohol and an acid: asp and tyrAlcohol and an acid: asp and tyrTwo acids: asp and gluTwo acids: asp and gluAlcohol and amine: ser and lysAlcohol and amine: ser and lysAlcohol and amide: ser and asnAlcohol and amide: ser and asn

4) 4) Covalent bondCovalent bond Created by chemical reactions that alter a Created by chemical reactions that alter a

polypeptide's structure during or after its polypeptide's structure during or after its synthesissynthesis

eg. Disulphide bond (strong linkage)eg. Disulphide bond (strong linkage) Protect protein structure from adverse changes Protect protein structure from adverse changes

in pH or salt concentrationsin pH or salt concentrations

d) Quaternary structured) Quaternary structure

Proteins consist of two or more polypeptide chains Proteins consist of two or more polypeptide chains aggregated into one functional macromoleculesaggregated into one functional macromolecules

Many proteins, esp those with high molecular weight Many proteins, esp those with high molecular weight are composed of several polypeptide chains.are composed of several polypeptide chains.

In proteins that consist of more than 1 polypeptide In proteins that consist of more than 1 polypeptide chain, each polypeptide is called chain, each polypeptide is called subunitsubunit

Polypeptide subunits assemble and held together by Polypeptide subunits assemble and held together by noncovalent interaction eg H bonding, hydrophobic noncovalent interaction eg H bonding, hydrophobic effect, electrostatic interactioneffect, electrostatic interaction

Loss of protein structureLoss of protein structure

Protein structure is sensitive to environment Protein structure is sensitive to environment factorsfactors

Many physical & chemical agents can disrupt Many physical & chemical agents can disrupt protein’s conformationprotein’s conformation

The process of structure disruption = The process of structure disruption = denaturationdenaturation

Denaturing conditions :Denaturing conditions :

Strong acid or baseStrong acid or base – changes in pH result in – changes in pH result in protonation of some protein side group, which protonation of some protein side group, which alter/disrupt hydrogen bonding & salt bridgealter/disrupt hydrogen bonding & salt bridge

Organic solventsOrganic solvents – water-soluble organic – water-soluble organic solvents eg. Ethanol interfere with solvents eg. Ethanol interfere with hydrophobic interactionhydrophobic interaction

DetergentsDetergents – these amphiphatic molecules – these amphiphatic molecules disrupt hydrophobic interaction causing disrupt hydrophobic interaction causing proteins to unfold into extended polypeptide proteins to unfold into extended polypeptide chains chains

(amphiphatic = contain nonpolar and polar (amphiphatic = contain nonpolar and polar components)components)

Reducing agentsReducing agents – eg. Urea, – eg. Urea, ββ--mercaptoethanol, will convert disulfide bridge mercaptoethanol, will convert disulfide bridge (S-S) to sulfhydryl group (SH)(S-S) to sulfhydryl group (SH)

urea disrupt H bond & hydrophobic interactionurea disrupt H bond & hydrophobic interaction

Heavy metal ionsHeavy metal ions – mercury (Hg – mercury (Hg++) and lead ) and lead (Pb(Pb2+2+) disrupt salt bridge by forming ionic bond ) disrupt salt bridge by forming ionic bond with negatively charge group.with negatively charge group.

Temperature changeTemperature change – as temp increase, the – as temp increase, the rate of molecular vibration increase. So weak H rate of molecular vibration increase. So weak H bond will be disrupt and protein will unfold.bond will be disrupt and protein will unfold.

Mechanical stressMechanical stress – stirring & grinding actions – stirring & grinding actions disrupt the delicate balance of forces that disrupt the delicate balance of forces that maintain protein strcuture.maintain protein strcuture.

eg. Foam formed when egg white is beaten eg. Foam formed when egg white is beaten vigorously contains denatured proteinvigorously contains denatured protein

Fibrous proteinsFibrous proteins

Fibrous protein exist as a long stranded Fibrous protein exist as a long stranded moleculesmolecules

Contain high proportions of secondary Contain high proportions of secondary structures ; structures ; αα-helices and -helices and ββ-pleated sheets-pleated sheets

Most are structural proteinMost are structural protein eg. eg. αα-keratin, collagen, silk fibroin-keratin, collagen, silk fibroin

αα-keratin-keratin Found in hair, wool, skin, horns, fingernails is Found in hair, wool, skin, horns, fingernails is

an an αα-helical polypeptides-helical polypeptides Each polypeptide has three domain :Each polypeptide has three domain :

- an amino terminal ‘head’- an amino terminal ‘head’

- a central rodlike - a central rodlike αα-helical domain-helical domain

- a carboxyl terminal ‘tail’- a carboxyl terminal ‘tail’ Two keratin polypeptides associate to form = Two keratin polypeptides associate to form =

coiled coil dimercoiled coil dimer

Two keratin polypeptides associate to form = Two keratin polypeptides associate to form = coiled coil dimercoiled coil dimer

Two antiparallel rows of these dimer form a Two antiparallel rows of these dimer form a supercoiled structure called a supercoiled structure called a protofilament protofilament (disulfide bond aid the formation of (disulfide bond aid the formation of protofilament)protofilament)

Hundreds of filaments, each containing 4 Hundreds of filaments, each containing 4 protofilaments form protofilaments form macrofibrilmacrofibril

Each hair cells (fiber) contain Each hair cells (fiber) contain several several macrofibrilsmacrofibrils

CollagenCollagen The most abundant protein in vertebratesThe most abundant protein in vertebrates Synthesized by Synthesized by

- connective tissue cells- connective tissue cells mostly found in mostly found in fibrous tissuesfibrous tissues such as : such as :

tendon, ligament and skin, in cornea, cartilage, tendon, ligament and skin, in cornea, cartilage, bone, blood vessels, the gutbone, blood vessels, the gut

Extremely strongExtremely strong

Collagen is composed of three Collagen is composed of three polypeptide helices that are twisted polypeptide helices that are twisted around each other to form a around each other to form a triple helixtriple helix (stabilized by (stabilized by hydrogen bondinghydrogen bonding))

The amino acid composition of collagen The amino acid composition of collagen is distinctiveis distinctive

- high content of glycine, proline and - high content of glycine, proline and lysinelysine

- very little amount of cysteine (unlike - very little amount of cysteine (unlike αα--keratin)keratin)

Silk fibroinSilk fibroin

Silk protein Silk protein - form spider webs, cocoon and nests- form spider webs, cocoon and nests- consist of the fibrous protein fibroin- consist of the fibrous protein fibroin

Considered to be Considered to be ββ-keratin -keratin - - polypeptide chains arranged in antiparallel polypeptide chains arranged in antiparallel ββ--pleated sheet comformationpleated sheet comformation

Its Its primary structureprimary structure mainly consists of the mainly consists of the amino acidamino acid sequence ( sequence (GlyGly--SerSer-Gly-Ala-Gly--Gly-Ala-Gly-Ala)Ala)nn

Globular proteinGlobular protein Globular protein have a Globular protein have a spherical shapespherical shape, ,

compact and water-solublecompact and water-soluble In their function, usually require them to bind In their function, usually require them to bind

precisely to other moleculesprecisely to other molecules Most enzyme are globular Most enzyme are globular Myoglobin & hemoglobinMyoglobin & hemoglobin are typical example are typical example

of globular proteinof globular protein Both are hemoprotein and each is involved in Both are hemoprotein and each is involved in

oxygen metabolismoxygen metabolism

Unlike fibrous proteins which only play a structural Unlike fibrous proteins which only play a structural function, globular proteins can act as:function, globular proteins can act as:

1) Enzymes, by catalyzing organic reactions taking 1) Enzymes, by catalyzing organic reactions taking place in the organism in mild conditions and with a place in the organism in mild conditions and with a great specificity. great specificity.

2) Messengers, by transmitting messages to regulate 2) Messengers, by transmitting messages to regulate biological processes. This function is done by biological processes. This function is done by hormones, i.e. insulin etc. hormones, i.e. insulin etc.

3) Transporters of other molecules through membranes 3) Transporters of other molecules through membranes 4) Stocks of amino acids. 4) Stocks of amino acids. 5) Regulatory roles are also performed by globular 5) Regulatory roles are also performed by globular

proteins rather than fibrous proteins. proteins rather than fibrous proteins.

a) Myoglobina) Myoglobin MyoglobinMyoglobin is an iron- and oxygen-transport is an iron- and oxygen-transport

protein found in the muscle tissue of vertebrates protein found in the muscle tissue of vertebrates in general and in almost all mammals.in general and in almost all mammals.

composed of a single polypeptide chain of 153 composed of a single polypeptide chain of 153 amino acid residues and has the ability to store amino acid residues and has the ability to store oxygen by binding it to an iron atom (heme)oxygen by binding it to an iron atom (heme)

It is found abundantly in the tissues of diving It is found abundantly in the tissues of diving mammals, e.g., the whale, the seal, and the mammals, e.g., the whale, the seal, and the dolphin. dolphin.

High concentrations of myoglobin in these High concentrations of myoglobin in these animals, allows them to store sufficient animals, allows them to store sufficient oxygen to remain underwater for long periods. oxygen to remain underwater for long periods.

A myoglobin polypeptide is comprised of 8 A myoglobin polypeptide is comprised of 8 separate right handed α-helices, that are separate right handed α-helices, that are connected by short non helical regions. connected by short non helical regions.

Each myoglobin molecule contains one heme Each myoglobin molecule contains one heme prosthetic groupprosthetic group

Each heme consist of porphyrin ring with FeEach heme consist of porphyrin ring with Fe2+2+ in the centerin the center

Free heme [FeFree heme [Fe2+2+] has a high affinity for O] has a high affinity for O2 2 and and

is irreversibly oxidized to form hematin [Feis irreversibly oxidized to form hematin [Fe3+3+]]

b) Hemoglobinb) Hemoglobin

Hemoglobin is a roughly spherical molecule Hemoglobin is a roughly spherical molecule found in red blood cellsfound in red blood cells

Function = transport oxygen from lung to Function = transport oxygen from lung to every tissues in the bodyevery tissues in the body

Composed - two Composed - two αα-chain -chain - two - two ββ-chain-chain

The protein contain four subunits, designated The protein contain four subunits, designated αα and and ββ. . Each subunit contain a heme group that Each subunit contain a heme group that bind with oxygenbind with oxygen