Post-translational modifications (PTMs) play an important role in modifying proteins after translation to achieve their functional forms. Key PTMs include: 1. Protein folding facilitated by chaperones enables proteins to achieve their native conformations. 2. Proteolytic cleavage activates proteins by cleaving propeptides or signal sequences. Enzymes like signal peptidase and procollagen peptidases are involved. 3. Covalent modifications like phosphorylation, acetylation, methylation regulate protein activity by modifying side chains. Over 150 types of covalent modifications exist.

1. Post translation modifications
D.INDRAJA

2. • Nascent polypeptides must be folded to their native conformations and
processed into its biologically active form
• Native conformation, occurs with the formation of appropriate hydrogen
bonds and vander Waals, ionic, and hydrophobic interactions.
• The disulfide bonds, if any, must be formed
• In the case of multisubunit proteins, the subunits must properly combine
• Once the protein has been synthesized by the ribosome from its
corresponding mRNA in the cytosol, many proteins get directed towards the
endoplasmic reticulum for further modification.
• Certain N and C terminal sequences are often cleaved in the ER after which
they are modified by various enzymes at specific amino acid residues. These
modified proteins then undergo proper folding to give the functional protein.
• Most of the proteins that are translated from mRNA undergo chemical
modifications before becoming functional in different body cells. The
modifications collectively, are known as post-translational modifications.

3. • Post translational modifications occurring at the peptide terminus of the
amino acid chain play an important role in translocating them across
biological membranes.
• These include secretory proteins in prokaryotes and eukaryotes and also
proteins that are intended to be incorporated in various cellular and organelle
membranes such as
• lysosomes,
• chloroplast,
• mitochondria and plasma membranes.
• Expression of proteins is important in diseased conditions. Post translational
modifications play an important part in modifying the end product of
expression and contribute towards biological processes and diseased
conditions.
• The amino terminal sequences are removed by proteolytic cleavage when
the proteins cross the membranes.
• These amino terminal sequences target the proteins for transporting them to
their actual point of action in the cell.

4. Importance of PTMs
• Play a crucial role in generating the heterogeneity in proteins.
• Help in utilizing identical proteins for different cellular functions in
different cell types.
• Regulation of particular protein sequence behavior in most of the
eukaryotic organisms.
• Play an important part in modifying the end product of expression.
• Contribute towards biological processes and diseased conditions.
• Translocation of proteins across biological membranes

5. Processes
Protein folding
Proteolytic Cleavage
Covalent Modification/chemical Modification
Protein Splicing: Inteins and Extein
Protein degradation

6. Protein folding
• For making linear polypeptide chain of amino acid (primary protein)
functional, certain folding and modifications take place in it which are called
post translational modifications.
History
• This was initially established by Christian Afinsen.
Principle
• The classic principle of protein folding is that all information required for a
protein to adopt the correct three dimensional conformation is provided by
its amino acid sequence.
• The proper folding of proteins with in cells is mediated by the activities of
other protein.
• Protein is only functional when it is in folded state. After translation formed
protein is linear so it do not have any function.
• For its folding certain enzymes and protein are required. So various
modifications occur in this nascent polypeptide chain

7. • Proteins must fold to assume their functional state. Folding can be
spontaneous or facilitated by proteins known as Chaperones
Chaperones
• Chaperones are heat shock proteins (discovered in response to heat shock).
• They facilitate & favour the interactions on the polypeptide surfaces to
finally give specific conformation of a protein.
• Chaperones can reversibly bind to hydrophobic regions of unfolded proteins
& folding intermediates.
• They can stabilize intermediates, prevent formation of incorrect
intermediates, and also prevent undesirable interactions with other proteins.
• All these activities of chaperones help the protein to attain compact &
biologically active conformation.
Types of chaperons
1. Hsp70 system
2. Chaperonin system

8. Hsp70 system
• This mainly consists of Hsp70 (70-kDa heat shock protein) & Hsp40 (40-
kDa Hsp).
• These proteins can bind individually to the substrate (protein) & help in the
correct formation of protein folding.
Chaperonin system
• This is a large oligomeric assembly which forms a structure into which the
folded proteins are inserted.
• The chaperonin system mainly has Hsp60 & Hsp10. i.e. 60 kDa Hsp and 10
kDa Hsp.
• Chaperonins are required at a later part of the protein folding process &
often work in association with Hsp70 system.
Action of chaperons during translation

9. • Enzymes that catalyze protein folding
• Protein disulfide isomerase- catalyzes disulfide bond formation between
cysteine residues of polypeptide chain. Most abundant in endoplasmic
reticulum.
• peptidyl prolyl isomerase – prefering of proline to cis form can be a limiting
factor for folding,so this enzyme convert cis form of proline to trans form
and help in folding.
The action of protein disulfide isomerase The action of peptidyl prolyl isomerase

10. Proteolytic Cleavage
• Proteolytic cleavage is the most common type of post translational
modification
• Proteolytic removal of their leading Met (or fMet) residue shortly after it
emerges from the ribosome
• Inactive precursors that are activated under proper conditionsby limited
proteolysis.
• Inactive proteins that are activated by removal of polypeptides are called
proproteins, whereas the excised polypeptides are termed propeptides.
• Conversion of trypsinogen and chymotrypsinogen to their active forms by
tryptic cleavages
• The formation of active insulin from the 84-residue proinsulin by the
excision of its internal 33-residue C chain

11. Collagen
• The N- and C-terminal propeptides of procollagen are respectively removed
by amino- and carboxy procollagen peptidases
• An inherited defect of amino procollagen peptidase in cattle and sheep
results in a bizarre condition, dermatosparaxis, that is characterized by
extremely fragile skin.
• Analogous disease in humans, Ehlers-Danlos syndrome VII

12. •Signal peptides are removed from nascent proteins by a signal peptidase
Proteins bearing a signal peptide are known as preproteins or, if they also
contain propeptides, as preproproteins.Once the signal peptide has passed
through the membrane, it is specifically cleaved from the nascent polypeptide
by a membrane-bound signal peptidase
•Both insulin and collagen are secreted proteins and are therefore synthesized
with leading signal peptides in the form of preproinsulin and preprocollagen.
Sequential proteolytic cleavages:
(1) The deletion of their initiating Met residue,
(2) The removal of their signal peptides and
(3) The excision of their propeptides

13. • Some proteins are synthesized as segments of polyproteins, polypeptides
that contain the sequences of two or more proteins. Eg proteins synthesized
by many viruses Specific proteases post-translationally cleave polyproteins
to their component proteins, presumably through the recognition of the
cleavage site sequences

14. Covalent Modification

15. Covalent Modification
• Proteins are subject to specific chemical derivatization, both at the functional groups
of their side chains and at their terminal amino and carboxyl groups.
• Such modifications, which may alter the activity, life span, or cellular location of
proteins
• Over 150 different types of side chain modifications, involving all side chains but
those of Ala, Gly, Ile, Leu, Met, and Val, are known
• acetylations, glycosylations, hydroxylations, methylations, nucleotidylations,
phosphorylations, and ADP-ribosylation
• Covalent modifications regulate the activity of enzymes andmany other
proteins.
• Most modifications are reversible, such as phosphorylation,methylation,
acetylation, and ubiquitination.
• Some modifications are not reversible, such as adding a lipid or sugar group.
• Modification occurs  Co-translationally: require particular aa sequence
contexts for recognition
• Post-translationally: require accessibility of the
target residues on the surface of the protein

16. MODIFICATION AMINO ACIDS THAT ARE MODIFIED
Acetylation Lys
Methylation Lys
Phosphorylation Ser,Thr,Tyr,Asp,His,Lys
Hydroxylation Pro,Lys
Carboxylation Glu( from γ-carboxy glutamic acid)
O-linked glycosylation Ser,Thr
N-linked glycosylation Asn
Acylation Ser,Thr.Cys
Myristoylation Gly
Palmitoylation Cys
Farnesylation Cys
Biotinylation Lys
ADP ribosylation At the nitrogen atom of His,Arg,Asn and
Lys or at carboxyl group of Glu

17. N-Acetylation
• This process involves the transfer of an acetyl group to nitrogen of Lys (K)
• It has both reversible and irreversible mechanisms.
• Acetylation helps in protein stability, protection of the N-terminus and the
regulation of protein-DNA interactions in the case of histones.
Methylation
• Protein methylation typically takes place on arginine or lysine amino acid
residues in the protein sequence. By (S-adenosyl methionine) methyl donor
• Methylation of histones, a type of DNA binding protein, can regulate DNA
transcription.

18. phosphorylation
• The hydroxyl group containing amino acids of proteins, namely serine,
threonine & tyrosine are subjected to phosphorylation.
• The phosphorylation may either increase or decrease the activity of the
proteins.
• A group of enzymes called protein kinases catalyses phosphorylation while
protein phosphatases are responsible for dephosphorylation.
• Many enzymes that undergo phosphorylation or dephosphorylation (e.g.
Glycogen synthase)
• It plays an important role in regulating many important cellular processes
such as cellcycle, growth, apoptosis (programmed celldeath) and signal
transduction pathways.)

19. Hydroxylation
• The biological process of addition of a hydroxyl group to a protein amino
acid is called Hydroxylation.
• Protein hydroxylation is one type of PTM that involves the conversion of –
CH group into –COH group and these hydroxylated amino acids are
involved in the regulation of some important factors called transcription
factors.
• Among 20, the two amino acids can be regulated these are proline and
lysine.

20. carboxylation
• Vitamin K dependent carboxylation of glutamic acid residues in certain clotting
factors is also a post-translational modification.
Glycosylation
• Glycosylation involves the enzymatic addition of saccharide molecules to
amino acid side chains.
• This can be of two types – N-linked glycosylation, which links sugar
residues to the amide group of aspargine and O-linked glycosylation, which
links the sugar moieties to the hydroxyl groups of serine or threonine.
• Suitable glycosyl transferase enzymes catalyze these reactions.
• Sugar residues that are attached most commonly include galactose,
mannose, glucose, Nacetylglucosamine, Nacetylgalactosamie as well as
fucose

21. N-myristoylation
It is the attachment of myristoyl group a 14- carbon saturated fatty acid (C14)
to a glycine in protein. • It is facilitated by N-myristoyltransferase (NMT) and
uses myristoyl-CoA as the substrate

22. S-palmitoylation
• It is addition of C16 palmitoyl group from palmitoyl-CoA of cys in protein
• Palmitoyl acyl transferases (PATs) enzyme favours this step.
• Reversed by thioesterases
S-prenylation
• Addition of a farnesyl (C15) or geranylgeranyl (C20) group to cys in
proteins.
• Enzyme involved in this reaction is farnesyl transferase (FT) or
geranylgeranyl transferases (GGT I and II).

23. Biotinylation
• The addition of biotin to the Lys in protein or nucleic acid
ADP-ribosylation
• ADP-ribosylation is the addition of one or more ADP-ribose moieties to
a protein.It is a reversible post-translational modification that is involved in
many cellular processes, including cell signaling, DNA repair, gene
regulation and apoptosis
Structure of ADP ribose

24. • Protein lipid-modifications including myristoylation, palmitoylation,
farnesylation, and prenylation, known for a long time, have been shown to
have a role in protein-membrane interactions, protein trafficking, and
enzyme activity.
ADP ribosylation on protein

25. Protein Splicing
• The primary translation product of a gene contains one or more short amino
acid sequences,called inteins that excise themselves from the nascent
polypeptide
• The sequences that are represented in the mature polypeptide are termed
exteins
• Inteins occur in both eukaryotic and prokaryotic polypeptides
• An intein has the ability to catalyze its own removal from primary
translation product
• The autocatalytic process of protein splicing involves bond transfer reactions
and no input energy is required
• It involves four steps
I. N-O shift
II. Transesterification
III. Asn cyclization
IV. O-N shift

26. N-O shift
• A typical intein begins with ser or cys and end in Asn. The first residue in
the C-extein is Ser, Thr or Cys. These residues often function as
nucleophiles.
• N-O shift is the transition of the peptide bond between the amino acid of
intein and N-extein in to ester or thio ester bond
• The transition depends on a nucleophile attack of the bond by the side chain
of the ser or cys residues at the terminal end of the intein (-OH or –SH
respectively)
• This reaction is termed N-O shift when the attacking atom is an oxygen and
N-S when the atom is sulphur
Transesterification
• In this step the side chain of the first residue of the C-extein attacks
the ester(or thio ester) bond at the amino end of the intein.
• Here too the attack is by a polar side chain of a Ser, The(both –OH) or
Cys(-SH).
• This leads to a transesterification and formation of thioester or ester
bond between N-extein and C-extein

27. Asn Cyclization
• Cyclization of Asn side chain leads to clevage of the peptide bond between
the intein and the C-extein (C- terminal splice junction)
• This reaction removes intein from the ligated exteins, which are linked
together via the ster bond
O-N shift
• This step of protein splicing is spontaneous
• The reverse N-O shift or N-S shift takes place and peptide bond formation
occurs between N and C –exteins
• Some inteins show sequence specific endonuclease activity also. Such
inteins cut DNA in the intein-minus gene at a specific point and allow a copy
of a DNA sequence coding intein to integrate. This event is similar to intron
homing and termed as intein homing

28. Protein splicing

30. Difference between Protein and RNA splicing

31. Protein Degradation
• Proteins that are defective for example, misfolded or destined for rapid
turnover are often marked for destruction by ubiquitination-the attachment
of chains of a small, highly conserved protein, called ubiquitin.
• Proteins marked in this way are rapidly degraded by a cellular component
known as the proteosome, which is a macromolecular, ATP- dependent,
proteolytic system located in the cytosol.
Diseases due to misfolding of protein
• The failure of a protein to fold properly generally leads to its rapid
degradation.
• Cystic fibrosis (CF) is a common autosomal recessive disease.
• Some cases of CF with mutations that result in altered protein (cystic
fibrosis transmembrane conductance regulator or in short CFTR).
• CFTR cannot fold properly, besides not being able to get glycosylated or
transported.
• CFTR gets degraded.

32. • Certain neurological diseases - due to cellular accumulation of aggregates of
misfolded proteins or their partially degraded products.
• The term prions (proteinous infectious agents) is used to collectively
represent them.
• Prions exhibit the characteristics of viral or microbial pathogens and have
been implicated in many diseases. e.g. Alzheimer's disease.