PROTEIN - CLASSIFICATIONPROTEIN - CLASSIFICATION

CLASSIFICATION

1. Simple soluble proteins - Trypsin, Ribonuclease2. Integral Membrane Proteins – Cytochrome c3. Conjugated proteins – Non amino acid part, called prosthetic group’ can

containi) Lipids eg – LDL - Lipoproteinii) Carbohydrate – Ig G - Glycoproteiniii) Phosphate – Casein - Phosphoproteiniv) Heme – Hb - Hemoproteinv) Metals – Ferritin, Calmodulin - Metalloprotein

PROPERTIES OF PROTEINS

• Proteins are made from a pool consisting of 20 standard amino acids.

• Amino acids are joined together by peptide linkages• Amino acids can be categorised as hydrophilic or hydrophobic

based on their R group• All amino acids are in α- configuration, and are L-forms• Peptides may be classified as tripeptide, oligopeptide or

polypeptide based on their length• All proteins have a polarity i.e., a N-terminal & C-terminal• Hydrolytic breakdown of proteins can occur

1. chemically (6N HCl) – rxn with FDNB to form 2,4-dinitrophenyl derivative of N-terminal; (CNBr) – acts at C-terminal of Met.

2. enzymatically (chymotrypsin) – C-terminal of amino acids F, Y, W. (trypsin) – N-terminal of amino acids F,Y,W.

PROTEIN - FUNCTION

PROTEIN STRUCTURE

ORDERS OF STRUCTURE

i) Primary Structure – Sequence of amino acids in a polypeptide chain.

ii) Secondary structure – the folding of short segments (3 – 30 aa) of polypeptide into geometrical shapes like α–helix, β- sheet & β- turn.

iii) Tertiary structure – 3D assembly of secondary structural units into larger functional polypeptide.

iv) Quaternary structure – many polypeptide chains arranged to form oligomeric

protein.

PRIMARY STRUCTURE

• Peptide bond has partial double bond characteristic and is not free to rotate.• Other bonds in vicinity are free to rotate & can take up new positions.

The N - Cα & Cα – C bonds can rotate to assume bond angles of φ and ψ respectively.

SECONDARY STRUCTURE

• Ramachandran plot drawn between φ and ψ values can predict the possible conformations adopted by amino acid side chains.

• Hydrogen bonds stabilize secondary structures.

TYPES OF SECONDARY STRUCTURETYPES OF SECONDARY STRUCTURE

TERTIARY STRUCTURE

• 3D conformation of entire polypeptide• Shows how secondary str assembles to form domains• Domain – a section of protein str sufficient to perform a particular chemical

or physical task like binding etc.

QUATERNARY STRUCTURE

• This structure defines the polypeptide composition of a protein.• Some proteins are made of >1 polypeptide; Individual subunits are denoted

as α,β,γ,δ etc. (eg Hb)

FACTORS THAT STABILIZE 3° & 4° STRUCTURE• Hydrophobic interactions or Vanderwaals interaction• Hydrogen bonds• Salt bridges – (between Glu & Lys)• Disulfide bonds - (between cys)

AMINO ACID BREAKDOWN

• GENERAL POINTS:• Proteins are degraded CONTINUOUSLY by proteases & peptidases to

aminoacids.• In humans, each day about 1-2% of total body protein, mainly muscle

proteins are degraded.• High rate of degradation is seen in pregnancy (uterine tissue) & starvation

(skeletal muscle).• Of liberated aa’s 75% is REUTILIZED.• Excess nitrogen forms ammonia that is converted to UREA, in humans

WAYS OF EXCRETING AMMONIA

MAIN REACTIONS OF AA CATABOLISM

REACTIONS OF AA CATABOLISM

I. TRANSAMINATION 1. Inter converts pairs of amino acids & keto acids. 2. Reversible and catalysed by amino transferases that require PALP as co-enzyme3. All aa’s except lys, thr, pro & hydroxyl pro can be transaminated.4. Rxn involves formation of a Schiff base intermediate

II. OXIDATIVE DEAMINATION

III. DEAMINATION

UREA CYCLE

MAIN FEATURES• Occurs in cytosol & mitochondria. • Reactions begin from NH3 formed by aminoacid breakdown• Citrulline formed leaves mitochondria to enter cytosol.• Rest of the rxns occur in cytosol, Fumarate formed enters mitochondria.

AMINO ACID

NUCLEIC ACIDS

INTRODUCTION:1. They are of 2 types DNA or RNA.2. DNA is made of nitrogenous bases adenine, guanine, cytosine and thymine.3. RNA is made of nitrogenous bases adenine, guanine, cytosine and uracil.4. GENE is a piece of DNA capable of forming a functional product either protein or RNA.5. Every cell typically has thousands of genes.6. RNA is of 3 major types rRNA – which is a component of Ribosomes, where proteins are synthesized mRNA – which carries the information to form protein tRNA – which acts as an adaptor molecule to translate info in mRNA into protein7. A nucleotide has 3 components; a nitrogenous base, pentose and phosphate. The nitrogenous base can be pyrimidines (C, T, U) or purines (A, G). 8. Some unusual bases like 5-methyl cytosine, 5-hydroxy methyl cytosine, pseudo uridine also occur. The pentose in DNA is deoxy ribose; and in RNA is ribose.9) Mononucleotides are linked by phosphodiester bonds in the 5` to 3` direction.

DNA - STRUCTURESTRUCTURE OF NUCLEOSIDES

General points and properties1. From X-ray diffraction data of

DNA, Chargaff found that Conc of A=T & Conc of G=C.

2. This led to proposal of double helical model, with pairing between purines &pyrimidines through hydrogen bonds.

3. In the normal form of DNA (B-DNA), the helix is right handed and the 2 strands are antiparallel.

FORMS OF DNA

RNAGeneral points and Differences from DNA(1) RNA contains ribose rather than deoxyribose (as in DNA).

(2) Instead of thymine, RNA contains uracil. Adenine, Guanine and Cytosine are common to DNA.

(3) RNA exists as a single strand. But, sometimes it can fold back on itself forming hairpins (eg) t RNA.

(4) Since RNA is a single strand molecule A # T & G # C.

(5) RNA can be hydrolysed by alkali to 2`,3`-cyclic diesters of mononucleotides unlike DNA.

TYPES OF RNA

1. mRNA • Very heterogeneous

in size and

stability. • In eukaryotes, mRNA is capped by

5-methyl guanosine

and has a poly A tail.• Cap helps in correct

recognition of start

site for translation,

tail prevents digestion by endonucleases.

2. tRNA• varies in length

from 74-95 nucleotides

• Atleast 20 diff tRNA exists for the 20 amino acids found in nature

3. rRNA• Large - 60 S

subunit – 28 S, 5.8 S and 5 S

rRNA• Small - 40 S

subunit - 18 S rRNA

DNA REPLICATION

MAIN FEATURES• DNA replication occurs in the nucleus.• It is semiconservative i.e daughter cell contains 1 parent strand that serves as template to form another new strand.• It begins at a specific origin and proceeds bidirectionally forming a replication fork.

DNA REPLICATION

SEMI – CONSERVATIVE MECHANISM

MESELSON – STAHL EXPERIMENT

DNA REPLICATION

THE MAIN ENZYME – DNA polymerase

DNA REPLICATION

EVENTS AT THE REPLICATION FORK• At the replication fork, 1 daughter strand is continuously formed (leading strand) while the other (lagging strand) is formed in fragments, 1000b long, called Okazaki fragments. • DNA pol is the main replicating enzyme. Eukaryotes contain 4 types of DNA pol.

DNA REPLICATION

A small ‘loop’ when introduced in the “lagging strand template” permits simultaneous synthesis of both daughter strands.

TRANSCRIPTION

MAIN FEATURES• DNA template is required – contains Initiation & Termination sites• ATP, GTP, CTP & UTP are required• New RNA’s are formed in 5’ → 3’ direction• In eukaryotes, it occurs inside nucleus and is catalysed by RNA POLYMERASE enzyme.• It has 2 parts (Core enzyme – α2ββ’) and σ subunit• There are 3 types of RNA pol.

TRANSCRIPTION

• Transcription in bacteria is simple to understand and consists of 3 steps – Initiation, elongation and termination.

INITIATION & ELONGATION• Initiation occurs from DNA regions called promoters, that are made of conserved sequences at specific positions.• It proceeds after formation of an “Open complex” between RNA pol & promoters• The σ subunit of RNA pol recognizes promoters and is immediately released after initiation is done.

TRANSCRIPTION

• Termination occurs at AT-rich sequence on the template strand. • It is usually preceded by atleast a couple of adjacent GC-rich regions.• These regions formation of a complementary GC rich seq in mRNA, resulting in a HAIRPIN LOOP.

Termination signal

Hairpin loop

POST TRANSCRIPTIONAL PROCESSING

• In prokaryotes, mRNA formed is immediately ready for protein synthesis• In eukaryotes, the mRNA formed in nucleus is very large & not fully processed.• It contains additional non-coding (interrupting) sequences called Introns. • The coding regions (exons) have to be cut and spliced together to form the mature mRNA.

POST TRANSCRIPTIONAL PROCESSING

• This process is called post-transcriptional processing and occurs inside nucleus in the spliceosome (made of small nuclear RNA’s & protein [snurps]). • The intron is removed as a lariat.• In eukaryotes rRNA and tRNA also undergo processing.

Lariat

GENETIC CODE

• FEATURES:

There are 20 amino acids in nature.If codon was 2 bases long 42 = 16If codon was 3 bases long 43 = 64

• Codon AUGAUG is always start codon (INITIATION CODON); It codes for Met.• 3 codons are stop codons (TERMINATION CODON); they are UAA, UGA & UAGUAA, UGA & UAG• Codons are degeneratedegenerate - Eg Met, Trp – has only 1 codon

Leu, Ser & Arg – has 6 codons

GENETIC CODE

• Recognition of codons in mRNA occurs through anticodon arm of tRNA. • Out of 64 codons, 61 codons give rise to 20 amino acids.• Hence for some aa. More than 1 code exists. This is called degeneracy.• It can be explained by WOBBLE WOBBLE HYPOTHESIS HYPOTHESIS proposed by Crick

WOBBLE HYPOTHESIS

Explains relationship between tRNA & mRNA.

MECHANISM OF WOBBLE

WOBBLE RULES:1. First 2 bases form strong base pairs and contribute to codon specificity2. First base of anticodon(5`) & last base of codon(3`) form a loose pairing, called wobble

base.3. For aa like Arg; many codons exist – Here, when first 2 bases are diffferent, a diff tRNA is

required.4. Hence it is seen that 32 tRNA’s required in total. (31 for aa & 1 for AUG).

PROTEIN SYNTHESISPROTEIN SYNTHESIS(Translation)(Translation)

INTRODUCTION

• Translation occurs in the cytoplasm

• Ribosomes are organelles that brings together mRNA, tRNA and aminoacids

to form proteins

• tRNA verifies Codon in mRNA and attaches the right amino acid

• The process is similar in Prok as well as Euk, with slight differences

• It occurs in 5 distinct steps viz., Activation of aa, Initiation,

Elongation, Termination & Posttranslational processing.

• Several cytoplasmic proteins help in the process. They are called

IF’s, EF’s & RF’s

1.ACTIVATION of amino acids – It achieves activation of –COOH gp & also the correct attachment of the aa with the corresponding tRNA.

2. INITIATION –

• mRNA bearing the code binds to both small

ribosome subunit (30S) and to initiating aa-tRNA,

directly at the ‘P’ site.

• Later, Large subunit (50S) binds to form the

initiation complex. GTP along with initiation factors

are required for this process.

3. ELONGATION –

Successive aa’s are

covalently attached

from their

corresponding tRNA.

It requires EF’s. It has 3

distinct steps:

a) Binding of incoming

aminoacyl-tRNA @ ‘A’

site.

b) Peptide bond

formation @ ‘A’ site

c) Translocation to ‘P’ site

4. TERMINATION & release – This is signaled by termination codons. Release factors are used in the process. • It leads to hydrolysis of the terminal peptidyl-tRNA bond.• Free polypeptide and the last tRNA is released.• Dissociation of 70S ribosome into 50S & 30S subunits.

Termination codons do not have corresponding tRNA or amino acid.

There are 3 RF’s used.RF-1 – recognizes termination codons UAG, UAARF-2 – recognizes termination codons UGA, UAARF-3 – function not clear, may dissociate ribosomal subunits.

5. Folding & POST-TRANSLATIONAL PROCESSING –

• Newly formed polypeptide must fold into a 3D form to attain complete biological

function.

• Some proteins also undergo enzymatic processing (removal of 1 or more aa,

addition of acetyl, phosphoryl, methyl, carboxyl or other groups, proteolytic

cleavage, attachment of oligosaccharides or prosthetic gps etc.

FATS

FATTY ACIDS & TG – Properties.• Amphipathic cpds in aqueous soln. • Fatty acids have very long hydrophobic alkyl chains that are surrounded by a layer of water molecules. • By clustering together as micelles the FA expose smallest possible hydrophobic surface area to water.

MICELLE

STRUCTURE – FATTY ACIDS & TG

• FA are carboxylic acids with hydrocarbons from FA are carboxylic acids with hydrocarbons from C4-C36.C4-C36. Some are saturated; others may Some are saturated; others may contain 1 or more double bonds. contain 1 or more double bonds. • The most commonly occurring FA have even no of carbon atoms of 12-24 carbons.The most commonly occurring FA have even no of carbon atoms of 12-24 carbons.• Double bonds usually occur after C9.Double bonds usually occur after C9.• Physical prop of FA is determined by the Physical prop of FA is determined by the lengthlength & & degree of unsaturationdegree of unsaturation of hydrocarbon of hydrocarbon chain. (longer the chain; and fewer the double bonds – lower the solubility).chain. (longer the chain; and fewer the double bonds – lower the solubility).

• Simple TG’s are tristearin,tristearin, tripalmitintripalmitin and trioleintriolein. But, most

naturally occurring TG’s are mixed.

• Since polar –OH gp of glycerol & -COOH gp of FA are linked by a

ester bond in TG, they are essentially nonpolar, hydrophobic mol

insoluble in water. They have lower sp.gravity than water.

TG - SYNTHESIS

• Animals synthesise and store Large amounts of TG in adiposetissue to use as fuel. • The first stage is formation of phosphatidic acid• Formation & breakdown of TG is regulated by hormones (Insulin favours formation)• 75% of FA formed from TG breakdown is re-esterified back to TG

FAT STORAGE, MOBILISATION & TRANSPORT

Fat storage:In plants – seeds (TG store)In animals – adipose tissue.Advantages:

1. Carbon atoms in FA are more reduced than in sugars. Oxidation yields >2 times energy gm/gm.2. TG are hydrophobic, hence unhydrated. Organisms that carry fat as fuel don’t carry extra wt of water of

hydration associated with polysacc.

FAT TRANSPORT FAT TRANSPORT

• TG stored in adipose tissue can be mobilized

by a

hormone-sensitive lipase.

• Such lipids are coated with a layer of

perilipins, a

family of proteins that restrict access to lipid

droplets, preventing untimely lipid

mobilization.

• Hormones epinephrine & glucagon,

secreted as a

result of low glc levels, activates adenylyl

cyclase

in membrane leading to cAMP pdn.

• cAMP activates hormone-sensitive lipase

causing

TG to breakdown to FA.

• FA is then transported to diff tissues like

skeletal muscle, heart & renal cortex bound to

albumin.

• Here they dissociate, enter cells and get

degraded.

LIPID MOBILIZATIONLIPID MOBILIZATION