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TOPIC 2: BIOMOLECULE 1DNA & PROTEIN

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Genetic information

Deoxyribonucleic acid

usually

Held together by

Double helix

Single stranded

Hydrogen bonds

Denaturation/Replication

maybe

B, A, Z

Ribonucleic acid

bases

Several forms

Anti parallel strands

Primary

Structural hierarchy

sequence

Secondary Tertiary

3D confirmation Supercoiling

Linked nucleotides

Transcribed to

Made ofMade of

e.g

sugar bases Phosphate ester link

AdenosineCytosineGuanineThymineUracil

has

Can become

Stabilized by

Exist in

has

has

Is three main

rRNA tRNA mRNA

Participate in

translation

Contains code for

Protein

Is synthesis of

e.g

Histones

Condense DNA in

chromatin

e.g

e.gisis

e.g

e.g

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DNA overview

• Hallmark of life – the ability to produce• The unique information for each individual

must be preserved and passed to progeny

• All life on earth uses nucleic acids for storage genetic information

• Except for viruses; all life use deoxyribonucleic acid (DNA) to store information

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Central dogma of Molecular Biology•Sequential genetic information transferred from DNA residue to synthesis protein

•DNA play essential role in heredity by serving as template for its replication.

•DNA cannot flow directly to synthesis a protein

•Genetic information from DNA is transferred to RNA through transcription

•The sequence of RNA is translated into a protein sequence

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Structure and components of the nucleotides

• Nucleic acid consist of nucleotide monomer

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OO=P-O O

Phosphate Group

NNitrogenous base (A, G, C, or T)

CH2

O

C1C4

C3 C2

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Sugar(deoxyribose)

Nucleic acids consist of repeating nucleotide that have phosphate ester, a pentose sugar, and a heterocyclic base.

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Nucleoside

• Base bound to pentose sugar

• Pentose sugar attached to ribose – ribonucleoside

• Pentose sugar attached to deoxyribose- deoxyribonucleosides

O

OH

N

N

NH2

O

CH2OP

O

O-

O-

deoxyctyidine monophosphate (dCMP)

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Types of nucleic acid

• DNA – Deoxyribonucleic acid• RNA – ribonucleic acid

O OHCH2

OHOH

HO HO O OHCH2

OH

ribose deoxyribose

(no O)

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Structure and component of the nucleotides

• Nucleotide also called as nucleic acid base• Base- refer to the nitrogen aromatic

compound• Nucleic acid base type: pyrimidine and purine• Pyrimidine – single ring• Purine – double ring

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Pyrimidine and Purine

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Nitrogen-Containing Bases

N

N

N

N

H

NH2

N

N

O

CH3

O

H

H

N

N

N

N

O

H

NH2

H

N

N

NH2

CH3

O

H

N

N

O

CH3

O

H

H

adenine (A) thymine (T)

guanine (G) cytosine (C) uracil (U)

Uracil generally only in RNAThymine generally only in DNA

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Nucleosides in DNA

Base Sugar NucleosideAdenine (A) Deoxyribose AdenosineGuanine (G) Deoxyribose GuanosineCytosine (C) Deoxyribose CytidineThymine (T) Deoxyribose Thymidine

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Nucleosides in RNA Base Sugar NucleosideAdenine (A) ribose AdenosineGuanine (G) ribose GuanosineCytosine (C) ribose CytidineUracil (U) ribose Uridine

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• Polymerization nucleotides form nucleic acids

• The phospodiester links the 5’OH of one residue and 3’OH of the next

• One end must terminate at in 5’OH, the other terminates at 3’OH.

O

N

N

NH2

O

CH2OP

O

O-

O-

OH

O

N

N

NH2

CH2OP

O

O-

OH

O

N

N

AMP

CMP

3

5

Β-glycosidic bond

3’-5’ Phosphodiester bonds

Formation of Nucleic Acid Structure

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Nucleic acid structure

Single letter represent individual base

Sequence of bases are unique and make each of us different!

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Double helix structure of DNA• Determination of double helix structure was

based on the X-ray diffraction patterns• Amount of T equal to A• Amount of G equal to C• Consist of 2 polynucleotide chain (we call it

DNA Strand) that wrapped to each other to form helix

• Chain run in antiparallel directions: 5’ to 3’ – sense strands 3’ to 5’ – antisense strands

• Sugar phosphate backbone – outer part• Bases pair is complementary:

A—T (2 H bond) G – C (3 H bond)

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Unwinding the helix

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Complementary base pairing

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DNA Sequence

Length of DNA sequence depends on organism:Bacteria e.g. Salmonella ~ 4Mb (4 million)Human ~ 3.4 Gb (billion)

• kb (= kbp) = kilo base pairs = 1,000 bp• Mb = mega base pairs = 1,000,000 bp• Gb = giga base pairs = 1,000,000,000 bp.

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DNA sequence• DNA sequence is obtained through Sequencing method• Sequence can be uploaded in NCBI database• Sequence of interest can also be found in the website

We will find time to review this webpage later…

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Denaturation of DNA

• A process by which double stranded DNA unwinds and separates into single stranded strands through the breaking of hydrogen bonding between the bases

• Can be achieved through heating the DNA in solution

• Complete denaturation- ~94°C

• Temperature needed depends on the base content of DNA; High G-C content will need a higher temperature. And why?

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Denaturation of DNA

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Renaturation of DNA

Reformation of complementary strands that were separated by heat by slow cooling process

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We will discuss on DNA compaction next week!

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PROTEIN 1:COMPOSITION AND

STRUCTURE

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Functional role of proteins in mammalian organism

• Catalysis in chemical transformations-enzymes• Transport –

Hemoglobin and myoglobin transport O2 in blood and muscle

Transferrin transport iron in blood• Metabolic control- enzymes involve in the process• Contraction – myosin and actin function in muscle

contraction• Matrix for bone and connective tissue – collagen and elastin

form the matrix of bone and ligament• Α-keratin- in hair and other epidermal tissue

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AMINO ACID COMPOSITION OF PROTEINS

• All different type of proteins are synthesized as polymers of only 20 amino acids

R Group- uniquely define each of amino acid

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R Groups of Amino Acids

Is used to classify amino acids:• Polar or non polar• Acidic or basic

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Polar uncharge

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Non polar hydrophobic

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Acidic

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Basic

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Abbreviations

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Amino acids can act as both acids and bases

CO2H – Can be deprotonated to become negative carboxylates (COO-) – cause acidity

NH2- – Can be protonated to become

positive α- ammonium groups (+NH3) – cause basic properties

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Amino acid as zwitterion

Basic groupAcidic group

H transfer

Zwitterion – a condition when amino acids are without charged groups on their side chain – no net charge; in solution- neutral

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Adding alkali to amino acid solution

+ [OH+]

Donate > [H+] to bind with +[OH+] NH3

+ become NH2 ( only –ve charge in COO- left)

Now this aa in negative charge!

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Adding acid to acid amino solution

+ [H+]

Now this aa in positive charge!

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Amino acid charge

General condition

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Amino acids are polymerized into peptides and proteins

• Isoelectric pH- the pH at which a molecule has no net charge – also called as isoelectric point (PI value)

• The PI- allow protein to be separated using electrophoresis, isoelectric focusing and ion exchange chromatography

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• TASK 1: Explain the function of plasma protein in diagnosis of animal disease

*Must include charge interaction and electrophoresis idea

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Two shape of proteins:• Fibrous protein• Globular protein

Fibrous protein Provide mechanical support Often assembled into large cables or threadse.g: α-keratin – major components of hair and nails

collagen – major components of tendons, skin, bones and teeth Involved in structure :tendon, ligaments, blood clots –

collagen and keratin Contractile protein in movement: muscle, microtubule

(cytoskleton, mitotic spindle, cillia, flagella)

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Globular protein• Usually water soluble, compact roughly spherical• Hydrophobic interior, hydrophilic surface• Globular protein include enzyme carrier and

regulatory protein• Most protein which move around (e.g albumin,

casein in milk)• Proteins with binding site:Enzymes, haemoglobin, immunoglobulin,

membrane receptor sites

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The peptide bond

• Peptide- short polymers of amino acid monomers linked by peptide bonds

• Polypeptide chain – longer peptide chain

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Hierarchy structure of protein

Primary structure (A.A sequence)

Secondary structure (α-helix and β-pleated sheet)

Tertiary structure (3-D structure formed by assembly of secondary structure)

Quaternary structure (structure formed by more than one polypeptide)

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Primary structure of proteins

Sequence of amino acid in polypeptide chainIs held together by peptide bondsTwo ends – N terminus and C terminus

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Secondary structure

• Local 3-D folding of the polypeptide chain in the protein

• Arrangement in space of the atoms in the peptide backbone

• Two type: α-helix and β-pleated sheet

α-helix β-pleated sheet

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• Forces involve:Strong – covalent bondWeak – hydrogen bond, electrostatic

interactions, hydrophobic effect

Secondary structure

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Tertiary structure

• 3D arrangement of all atoms in the proteins, including those in side chains and in prosthetic group

• Describes the folding and other contortions of a polypeptide chain that result from the molecular interactions among the R groups of the different amino acids

• The folding is sometimes held together by strong covalent bonds (cystein-cystein disulphide bridge)

• 3-D structure is determined through X-tray crystallography• Now can be predicted using bioinformatic technique

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Forces involved in tertiary structure

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Quaternary structure

• Arrangement of polypeptide chains in a multi chain protein

• The chain is called subunit• Subunit must be in non covalent association, maybe

connected by disulfide bonds• Not all protein have this structure• E.g.

chymotrypsin contains 3 polypeptide joined together by interchain disulfide bonds Hemoglobin – bohr effect

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DENATURATION AND REFOLDING• Non covalent interactions that

maintain 3-D structure of protein are weak – can be disrupted easily

• Unfolding a protein (i.e. disruption of tertiary structure)- denaturation

• Denaturation and reduction of disulfide bonds- happen when complete distruction of tertiary structure is desired

• Disruption process can be recovered - refolding

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Denaturation

Can be achieved in two ways:

1. Heat – increase temperature trigger vibration in molecules – when energy become great enough can disrupt the tertiary structure

2. At extreme high or low pH- some charges will be missing- so electrostatic interactions that normally stabilize the native and active form of protein are drastically reduced

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Denaturation3. Detergents – binding of detergents (e.g. sodium

dodecyl sulfate)- can disrupt hydrophobic interaction

charged detergent – disrupt electrostatic interactions

urea / guanidine hydrochloride – form hydrogen bonds with protein that stronger than internal hydrogen bond

B- mercaptoethanol – reduce disulfide bridge to 2 sulfuhydryl groups

#what do they use to straighten your curly hair?

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Relationship between protein structure and its function

• Protein structure determines protein function• Denaturation or inhibition may change protein

structure - will change its function• Coenzyme and co factor may enhance the

protein’s structure

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Protein folding• A process in which a

polypeptide folds into a specific, stable, functional 3-D structure

• In order to carry out their function (e.g. enzymes or antibodies), protein must take on a particular shape, also known as ‘fold’ – from 1° to 3°

• Thus protein are amazing machine! Before they do their work, they assemble themselves! This self- assembly is called ‘folding’

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The importance of correct folding

• Primary structure carry all the information needed to produce correct tertiary structure – but the process it self sometimes not straight fwd and trickier

• Protein dense environment cell – protein may fold incorrectly as they produced

• Or they may begin to associate with other protein before they complete their own folding

• In euk – proteins may need to remain unfolded long enough to be transported across the membrane

• Correctly folded – usually soluble in aqueous cell environment or attached in membrane

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The importance of correct folding

• Folding incorrectly – may interact with other proteins and form aggregates (Accumulate and clump together )

• Fail to do so – ineffective use of protein or producing toxic protein! – lead to protein folding disease

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• Occur because hydrophobic regions that should be buried in side the protein remain exposed and interact with other hydrophobic regions of other molecules Hydrophobic chain –

red color – interior Hydrophilic chain –green color –exterior

The importance of correct folding

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Protein folding chaperone

• Chaperone – special protein that help in correct and timely folding protein

• Prevents protein form associating with another protein or prevent it from associating with itself in inappropriate way

• E.g. of chaperone – hsp 70 (70 kilodalton heat shock protein)

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Chaperon helps in formation of hemoglobin

• Hb – consist of α globin and β globin chain

• Produced by α and β globin gene• There are 2 α globin gene for one β

globin gene – thus there is excess of α globin chain – aggregate among themselves damaged RBC – thallesemia – useless form of hemoglobin

• Therefore, α globin chain need to kept from aggregating – so they are enough in to complex with β globin chain

• With help from chaperone – α hemoglobin stabilizing protein – prevent α globin chain from causing damaged to RBC and help to deliver them to β globin chain

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TASK 2 – DISCUSS THE EXAMPLE OF PROTEIN FOLDING DISEASE BY STATING THE MECHANISM