6. A LITTLE BACKGROUND: PROTEINS
Plays a very significant role in the
structural and functional organization of
any cell.
Translation is the final stage of gene
expression and synthesize the
immature protein.
Many transmembrane or secretory
preproteins have an N-terminal signal
peptide.
A signal peptide is recognized by a SRP
that binds the translocon.
6
7. WHAT THE POST TRANSLATIONAL
MODIFICATION IS???
Post translational modification (PTM) is the
chemical modification of a protein after its
translation.
OR
The chemical modifications that take place
at certain amino acid residues after the
protein is synthesized by translation are
known as post-translational modifications.
These are essential for normal functioning
of the protein.
PTMS occur mostly in E.R and golgi
apparatus.
7
8. Why PTM is necessary???
Stability of protein
Biochemical activity (activity regulation)
Protein targeting (protein localization)
Protein signaling (protein-protein
interaction,cascade amplification)
8
10. 1,000,000 proteins out of only 30,000
genes???
•Splicing Variants
In eukaryotic cells, likely 6-8
proteins/gene.
• Post-translational modification
22 different forms of antitrypsin
observed in human plasma.
Post-translational modifications are key
mechanisms to increase proteomic
diversity and regulate
cellular activity.
10
13. 13
• Specific and well-regulated
• Enzymatic and Non-Enzymatic
• Examples:
Removal of signal leader peptide by signal peptidase
Precursor protein → mature protein (Insulin)
Zymogen → active enzyme
Trypsinogen → Trypsin
Prohormone → Hormone
Modification Involving Peptide Bonds Cleavage
(Limited Proteolysis)
13
20. How does Histone Acetylation
promote Transcription
• Acetylation neutralizes the positively charged Lys residues on histones
and thus reduces the interactions of histones with DNA.
Ac
BD
H4
5/8 12/16
TAFII250
Ac
BD
20
21. • Citrullination:
The conversion of arginine to citrulline.
Arginine Citrulline
• Deamination:
The conversion of glutamine to glutamic acid or
asparagine to aspartic acid.
PTMs involving changing the chemical
nature of amino acids
21
PAD
22. PROTEIN
FOLDING
• Physical process leading from an unfolded
polypeptide chain to a functional protein with a
definite structure.
• Minimizing the number of hydrophobic side-
chains exposed to water is an important driving
force.
• The native state is the most stably folded
form.
POST TRANSLATION MODIFICATION
23. 23
Protein Folding
The folding process
depends on the solvent , the
salts concentration , the pH,
the temperature and
molecular chaperones.
Chaperones are proteins
that facilitate the folding of
other proteins without being
part of assembled complex .
24. In vivo Protein folding in absence or presence of
Chaperones
Properly Folded
proteins
Improper folding
and Aggregation
Refolding
Proteosome
Degradation
25. SUBUNIT
AGGREGATION
Multimeric proteins are assembled in
the ER.
Some folded protein chains (subunits)
must aggregate with other subunits to
form quaternary structure.
Such multi-subunit proteins include
many of the most important enzymes
and transport proteins in the cell.
25
27. • Intramolecular process catalyzed entirely
by amino acid residues contained in the
intein.
• No coenzymes or sources of metabolic
energy.
• Involves bond rearrangements rather
than bond cleavage followed by
resynthesis.
• Converts Inactive protein precursor to
biologically active protein.
PROTEIN SPLICING
28. 28
What the INTEINS are
• An intein is a segment of
a protein that is able to
excise itself and join the
remaining portions (the
exteins) with a peptide
bond.
• Found in bacteria
eukaryotes, archaea and
viruses.
30. 30
Step 1
Formation of a linear ester
intermediate by NO or NS
acyl rearrangement
involving the first
nucleophilic amino acid
residue at the N-terminal
splice junction and the final
residue of the N-extein.
Step 2
Formation of a branched
ester intermediate by the
attack of the first
nucleophilic residue of the
C-extein on the linear ester
intermediate.
Step 3
Cyclization of the last
residue(Asn) of the intein,
cleaves apart the peptide
bond between the intein
and the C-extein, resulting
in a free intein segment with
a terminal cyclic imide.
Step 4
Spontaneous
rearrangement of the ester
linkage between the
ligated exteins to the
more stable amide bond.
The last step is
spontaneous and
irreversible.
The first three steps are
catalyzed by the intein.
What actually the Mechahanism of Splicing
is???
32. Regulation of :
1. Enzymatic activity
2. Half life of proteins
3. Interaction with other molecules
4. Subcellular localization of proteins
Rapid Purification of Target proteins.
In vitro Intein mediated Protein
Purification
What are the Applications ???
38. Proteome array containing
potential substrates for
phosphorylation
Kinase
enzyme
[g-33P] ATP
solution
Protein
substrate
Kinase enzyme
[g-33P]
ATP
ADP
Ser
Phosphorylated
protein
Ser
Microarray-based Detection Techniques for
PTMs
• Protein MicroarraysExample
40. 40
Does Post-translational Modification Occurs
in Prokaryotes???
Chemical modifications e.g. Phosphorylation
Classical system
Two-component system
PTS system
Signal peptide cleavages
Cleavages of N-terminal f- methionine residues
Protein Splicing
41. 41
SUMMARY
• PTM is the chemical modification of a protein after its translation.
• PTMs are key mechanisms to increase proteomic diversity and
regulate cellular activity.
• PTMs include modifications of peptide bonds, amino acids, subunit
aggregation and protein folding.
• Protein splicing is intramolecular process catalyzed entirely by amino
acid residues contained in the intein.
• PTMs can be detected by Gel based detection techniques, MS
techniques and Microarray based detection techniques.
41
Protein translation: The process by which the mRNA template is read by ribosomes to synthesize the corresponding protein molecule on the basis of the three letter codons, which code for specific amino acids.
SRP: Signal Recognition Particle.
N terminal signal peptide is also known as Leader sequence. It is hydrophobic amino acid chain present in nascent state configuration of proteins. After formation, a protein moves from ribosome to ER where SRP recognizes the signal peptide. Protein passes through a channel of proteins called TRANSLOCON present on the ER membrane and in prokaryotes it is present on plasma membrane. Here in ER the signal peptide is cleaved by signal peptidase.
Stability of protein is done mainly by folding process in which nascent state is converted into native state of protein.
Biochemical activity regulation means proteins are activated inside the cell whenever they are required otherwise they remain inactive. This is only possible by post translational modifications.
N-glycosylation is the addition of carbohydrate occurs on amino group of amino acids.
O-glycosylation is the addition of carbohydrate occurs on hydroxyl group of amino acids.
GPI: glycosyl phosphatidyl inositol
Enzymatic Cleavage by the use of enzymes i.e. Trypsin, Chymotrypsin
Non-enzymatic cleavage by the use of chemicals or heat i.e Cyanogen Bromide is often used to cleave the peptide bond after a methionine.
1st step: disulphide bond formation
2nd step: Removal of leader sequence by signal peptidase.
3rd step: Cleavage of C peptide (C for connecting) and formation of active fragment insulin.
The peptide bond of Proline with other amino acids is in cis form. During folding this cis bond is converted to trans form by an enzyme prolyl isomerase.
Prenylation: Addition of isoprenoid units. It is also called lipidation.
ADP Ribosylation: Addition of one or more ADP ribose moieties to a protein.Cholera toxin ADP-ribosylates G proteins causing massive fluid secretion from the lining of the small intestine, resulting in life-threatening diarrhea.
Phosphorylation is done by protein kinases and it mainly activates the proteins like in case of signaling proteins. While phosphatases dephosphorylate the protein mainly resulting in inactivation of protein.
Ubiquitin is small 8.5 kDa protein that attaches preferably at lusine residue by help of E3 ligase. i.e. ubiquitination of histone protein
PAD: peptidyl arginine deiminase
There are two classes of chaperons:
Molecular chaperones that only prevent improper folding.
Chaperonins or Heat shock proteins which are also involved in proper folding of a protein.
The DNA with coding regions for inteins are transcribed into mRNA and translated into a premature protein. A protein is not fully mature until a intein is excised from the protein.
DOD = dodecapeptide
The type of the splicing proteins is categorized into four classes: maxi-intein, mini-intein, trans-splicing intein, and alanine intein.
The maxi-inteins are N- and C-terminal splicing domains containing an endonuclease domain.
The mini-inteins are typical N- and C-terminal splicing domains; however, the endonuclease domain is not present.
The trans-splicing inteins are split inteins which are divided into N-termini and C-termini.
Alanine inteins have the splicing junction of an alanine instead of a cysteine or a serine, in both of which the protein splicing occurs.
Like introns within an RNA precursor, inteins are found within a mRNA strand. Just as introns are connected to RNA splicing, inteins are connected to protein splicing.
One of the best understood posttranslational modifications is the reversible phosphorylation of proteins at serine, threonine, or tyrosine residues. These phosphoamino acids are relatively stable in acidic solutions.
2. Immunoblotting: This process, also known as Western blotting, is a commonly used analytical technique for detection of specific proteins in a given mixture by means of specific antibodies to the given target protein.
Protein mixture containing phosphorylated as well as other unmodified proteins can be separated by a suitable electrophoresis technique. SDS-PAGE and two dimensional gel electrophoresis are most commonly used for protein separation. Two-dimensional gel electrophoresis (2-D electrophoresis) separate proteins in two steps, according to two independent properties: the first-dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI); the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (MW).
The separated protein bands are then blotted onto a nitrocellulose membrane. These membranes are then probed by means of specific anti-phospho-amino acid antibodies that specifically bind to proteins having phosphorylation at a particular amino acid residue. This binding interaction can then be detected by means of suitably labeled secondary antibodies or by autoradiography using a radioactive probe.
MALDI-TOF-MS: A mass spectrometry instrument that produces charged molecular species in vacuum, separates them by means of electric and magnetic fields and measures the mass-to-charge ratios and relative abundances of the ions thus produced. In MALDI-TOF-MS, the ion source used is MALDI. The Time-of-Flight mass analyzer correlates the flight time of the ion from the source to the detector with the m/z of the ion.
The modified protein of interest is digested into smaller peptide fragments using a suitable enzyme like trypsin. This digest is then mixed with a suitable organic matrix such as a-cyano-4-hydroxycinnamic acid, sinapinic acid etc. and then spotted on to a MALDI plate.
The target plate containing the spotted matrix and analyte is placed in a vacuum chamber with high voltage . The laser energy gets absorbed by the matrix and is transferred to the analyte molecules which undergo rapid sublimation resulting in gas phase ions. These ions are accelerated and travel through the flight tube at different rates. The lighter ions move rapidly and reach the detector first while the heavier ions migrate slowly. The ions are resolved and detected on the basis of their m/z ratios and a mass spectrum is generated.
Protein microarrays: These are miniaturized arrays normally made of glass, onto which small quantities of many proteins can be simultaneously immobilized and analyzed.
Potential substrates for protein phosphorylation are immobilized on a suitably coated array surface. To this, kinase enzyme and gamma P-32 labeled ATP are then added and the array is incubated at 30oC. The phosphorylation reaction occurs at those sites containing proteins that can be modified.
After sufficient incubation, excess unbound ATP and enzyme are washed off the array surface. Detection is carried out by means of autoradiography. The radioactive emissions from the phosphate label present at the phosphorylated protein sites strike the film. Upon development, the positions at which phosphorylation has occurred can be clearly determined.
“Classical system” utilizes NTPs as phosphoryl donors and leads to protein modification at Ser/Thr or Tyr residues.
“Two-component system” requires first a sensor kinase which autophosphorylates at a His residue at the expense of ATP, then in turn a response regulator is modified at an Asp residue and thereafter induces a metabolic change within cell.
“PTS system” use PEP to generate a phosphoryl group which is passed down a chain of several proteins and finally transferred to a sugar.
Cleavages of N-terminal f- methionine residues: the formyl group of f-met is removed by deformylase. The initiating met residue of some polypeptides may also be removed by methionine aminopeptidase.