Contents Introduction . Modifications Effects of PTMs Types of
PTMs Protein Splicing Applications Detection Techniques Summary
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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
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
Why PTM is necessary??? Stability of protein Biochemical
activity (activity regulation) Protein targeting (protein
localization) Protein signaling (protein-protein
interaction,cascade amplification) 8
9
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
Post-translational modification Modification Involving Peptide
Bonds Modification of amino acids Subunit aggregation Protein
folding and chaperones 11 Protein Splicing
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
14 iIn vivo conversion of preproinsulin to Insulin 14 (103
amino acids) (51 amino acid)
15 Ser esters Cys thioesters Asp or Asn isoaspartate Prolyl
peptide cis-trans isomerization by prolyl isomerase Modification
Involving Peptide Bond Isomerization (Intramolecular) 15
Modification of amino acids 16
17
Different types of PTMs & their modification sites
Phosphorylation Glycosylation Acylation Alkylation Hydroxylation
Pro, Lys Ser, Thr, Tyr Asn, Ser, Thr Asn, Gln, Lys Lys, Arg 18
Histone Modification Different modifications occur on specific
residues to perform specific regulatory functions. 19
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
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
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 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 .
In vivo Protein folding in absence or presence of Chaperones
Properly Folded proteins Improper folding and Aggregation Refolding
Proteosome Degradation
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
Haemoglobin A 26
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 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.
29 How Inteins look like??? Split intein Mini intein Maxi
intein
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???
Analogy to RNA Splicing
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 ???
Direction of migration Anode Cathode - + Buffer Acrylamide gel
Sample loading Protein mixture SDS-PAGE 2-D Electrophoresis
Proteins focused on IPG strip Direction of migration Completed
stained gels Gel-based Detection Techniques
ImmunoblottingExample
35 Completed gels Nitrocellulose sheetBlotting Specific
phospho- tyrosine antibodies added Detection using labeled
secondary antibodies Proteins phosphorylated at Tyr residues
Proteins phosphorylated at Tyr residues Immunoblotting
36 PTM modified protein of interest Trypsin digestion Protein
Matrix 196 well MALDI Plate Digested protein MS-based Detection
Techniques for PTMs MALDI-TOF-Mass SpectrometryExample Digestion
and Sample Spotting
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
Proteome array Washing Phosphorylated proteins Detection-
Autoradiography film 33P 33P 33P 33P33P Developed image Radioactive
emissions Protein Microarrays
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 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