PROTEIN - CLASSIFICATION
PROTEINS
SIMPLE SOLUBLE
INTEGRAL
MEMBRANE
CONJUGATED
CLASSIFICATION
1.
2.
3.
Simple soluble proteins - Trypsin, Ribonuclease
Integral Membrane Proteins – Cytochrome c
Conjugated proteins – Non amino acid part, called prosthetic group’ can
contain
i) Lipids eg – LDL
- Lipoprotein
ii) Carbohydrate – Ig G - Glycoprotein
iii) Phosphate – Casein
- Phosphoprotein
iv) Heme – Hb
- Hemoprotein
v) Metals – Ferritin, Calmodulin - Metalloprotein
PROPERTIES OF PROTEINS
•
•
•
•
•
•
•
1.
2.
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
chemically (6N HCl) – rxn with FDNB to form 2,4-dinitrophenyl derivative of Nterminal; (CNBr) – acts at C-terminal of Met.
enzymatically (chymotrypsin) – C-terminal of amino acids F, Y, W. (trypsin) – Nterminal 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
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•
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 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
II. OXIDATIVE DEAMINATION
I. TRANSAMINATION
1. Inter converts pairs of amino
acids & keto acids.
2. Reversible and catalysed by
amino transferases that require
PALP as co-enzyme
3. All aa’s except lys, thr, pro &
hydroxyl pro can be transaminated.
4. Rxn involves formation of a Schiff
base intermediate
III. DEAMINATION
UREA CYCLE
AMINO ACID
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.
NUCLEIC ACIDS
INTRODUCTION:
1.
2.
3.
4.
They are of 2 types DNA or RNA.
DNA is made of nitrogenous bases adenine, guanine, cytosine and thymine.
RNA is made of nitrogenous bases adenine, guanine, cytosine and uracil.
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
protein
7. 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 - STRUCTURE
STRUCTURE OF NUCLEOSIDES
General points and properties
1. 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 (BDNA), the helix is right handed
and the 2 strands are antiparallel.
FORMS OF DNA
RNA
General 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 = 16
If codon was 3 bases long 43 = 64
• Codon AUG is always start codon (INITIATION CODON); It codes for Met.
• 3 codons are stop codons (TERMINATION CODON); they are UAA, UGA & UAG
• Codons are degenerate 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
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 specificity
2. 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 SYNTHESIS
(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, UAA
RF-2 – recognizes termination codons UGA, UAA
RF-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 C4-C36. Some are saturated; others may
contain 1 or more double bonds.
• The most commonly occurring FA have even no of carbon atoms of 12-24 carbons.
• Double bonds usually occur after C9.
• Physical prop of FA is determined by the length & degree of unsaturation of hydrocarbon
chain. (longer the chain; and fewer the double bonds – lower the solubility).
• Simple TG’s are tristearin, tripalmitin and triolein. But, most
naturally occurring TG’s are mixed.
• Since polar –OH gp of glycerol & -COOH gp of FA are
ester bond in TG, they are essentially
linked by a
nonpolar, hydrophobic mol
insoluble in water. They have lower sp.gravity than water.
TG - SYNTHESIS
• Animals synthesise and store
Large amounts of TG in adipose
tissue 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
LIPID MOBILIZATION
• 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.