Translation: Protein synthesis

By – Dr. Mafatlal M. Kher
Unit: 2 Translation

DNA
Transcription
RNA
Translation
PROTEIN
Central
Dogma
of
Life
2

Importance of Proteins
“In my theatre, the nucleic acids write the script but the
enzymes (proteins) do the acting”
 Arthur Kornberg (Nobel Prize Winner Physiology/Medicine, 1959)
3

Translation
Synthesis of
Protein from mRNA
4

Translation
It is the mechanism by which the triplet
base sequences of m-RNA molecules are
converted into a specific sequence of amino
acids in a polypeptide-chain. It occurs on
ribosomes.
5

Why translation?
 The synthesis of proteins consumes more of a cell’s energy than any other
metabolic process.
 In turn, proteins account for more mass than any other macromolecule of
living organisms.
 They perform virtually every function of a cell, serving as both functional
(e.g., enzymes) and structural elements.
 The process of translation, or protein synthesis, the second part of gene
expression, involves the decoding by a ribosome of an mRNA message into
a polypeptide product.
6

The Genetic Code
 Translation of the mRNA template converts nucleotide-based genetic information
into the “language” of amino acids to create a protein product.
 A protein sequence consists of 20 commonly occurring amino acids.
 Each amino acid is defined within the mRNA by a triplet of nucleotides called
a codon.
 The relationship between an mRNA codon and its corresponding amino acid is
called the genetic code.
7

The Genetic Code
 The three-nucleotide code means that there is a total of 64 possible combinations
(43, with four different nucleotides possible at each of the three different positions
within the codon).
 This number is greater than the number of amino acids and a given amino acid is
encoded by more than one codon. This redundancy in the genetic code is
called degeneracy.
 Typically, whereas the first two positions in a codon are important for determining
which amino acid will be incorporated into a growing polypeptide, the third
position, called the wobble position, is less critical.
 In some cases, if the nucleotide in the third position is changed, the same amino
acid is still incorporated.
8

The Genetic Code
 Whereas 61 of the 64 possible triplets code for amino acids, three of the 64
codons do not code for an amino acid; they terminate protein synthesis, releasing
the polypeptide from the translation machinery.
 These are called stop codons or nonsense codons. Another codon, AUG, also
has a special function.
 In addition to specifying the amino acid methionine, it also typically serves as the
start codon to initiate translation.
 The reading frame, the way nucleotides in mRNA are grouped into codons, for
translation is set by the AUG start codon near the 5′ end of the mRNA.
 Each set of three nucleotides following this start codon is a codon in the mRNA
message.
9

The Genetic Code
 The genetic code is nearly universal.
 With a few exceptions, virtually all species use the same genetic code for
protein synthesis, which is powerful evidence that all extant life on earth
shares a common origin.
 However, unusual amino acids such
as selenocysteine and pyrrolysine have been observed in archaea and
bacteria.
 In the case of selenocysteine, the codon used is UGA (normally a stop
codon).
 However, UGA can encode for selenocysteine using a stem-loop structure
(known as the selenocysteine insertion sequence, or SECIS element),
which is found at the 3′ untranslated region of the mRNA.
 Pyrrolysine uses a different stop codon, UAG. The incorporation of
pyrrolysine requires the pylS gene and a unique transfer RNA (tRNA) with a
CUA anticodon.
10

The Genetic Code
This figure shows the genetic
code for translating each
nucleotide triplet in mRNA into an
amino acid or a termination signal
in a nascent protein. The first
letter of a codon is shown
vertically on the left, the second
letter of a codon is shown
horizontally across the top, and
the third letter of a codon is
shown vertically on the right.
11

THINK ABOUT IT
 How many bases are in
each codon?
 What amino acid is coded
for by the codon AAU?
 What happens when a stop
codon is reached?
12

The Protein Synthesis Machinery
 In addition to the mRNA template, many molecules and macromolecules
contribute to the process of translation.
 The composition of each component varies across taxa; for instance,
ribosomes may consist of different numbers of ribosomal RNAs (rRNAs)
and polypeptides depending on the organism.
 However, the general structures and functions of the protein synthesis
machinery are comparable from bacteria to human cells.
 Translation requires the input of an mRNA template, ribosomes, tRNAs,
and various enzymatic factors.
13

Ribosomes
Ribosomes are a cell structure that
makes protein. Protein is needed
for many cell functions such as
repairing damage or directing
chemical processes. Ribosomes
can be found floating within the
cytoplasm or attached to the
endoplasmic reticulum.
The location of the ribosomes in a
cell determines what kind of protein
it makes. If the ribosomes are
floating freely throughout the cell, it
will make proteins that will be
utilized within the cell itself. When
ribosomes are attached to
endoplasmic reticulum, it is referred
to as rough endoplasmic reticulum
or rough ER. Proteins made on the
rough ER are used for usage inside
the cell or outside the cell.
14

Transfer RNA (tRNA)
 tRNA is a type of RNA molecule that helps decode a messenger
RNA (mRNA) sequence into a protein.
 tRNAs function at specific sites in the ribosome during
translation, which is a process that synthesizes a protein from an
mRNA molecule.
 Proteins are built from smaller units called amino acids, which
are specified by three-nucleotide mRNA sequences called
codons. Each codon represents a particular amino acid, and
each codon is recognized by a specific tRNA.
 The tRNA molecule has a distinctive folded structure with
three hairpin loops that form the shape of a three-leafed clover.
One of these hairpin loops contains a sequence called the
anticodon, which can recognize and decode an mRNA
codon.
 Each tRNA has its corresponding amino acid attached to its
end. When a tRNA recognizes and binds to its corresponding
codon in the ribosome, the tRNA transfers the appropriate
amino acid to the end of the growing amino acid chain.
 Then the tRNAs and ribosome continue to decode the mRNA
molecule until the entire sequence is translated into a protein.
Amino acid
attachment
site
Intramolecular
base pairing
Anticodon
Codon
mRNA 5’ 3’
15

Transfer RNA (tRNA): Extra information
 The shortest RNA molecule in the cell, consisting of about 76 to 86
nucleotides.
 tRNAs carry amino acids to the ribosomes during protein synthesis.
 The majority of cells have 40 to 61 types of tRNAs because most of
the 61 sense codons have their own tRNA in the eukaryotic cytosol.
 The tRNAs, which accept the same amino acid are known as
isoaccepting tRNAs.
 In the human mitochondria, there are only 22 different tRNAs and in
plant chloroplasts, about 30.
 tRNA is frequently called an adaptor molecule because it adapts the
genetic code for the formation of the primary structure of protein. Rarely
(ca. 1/3000), a tRNA is charged with the wrong amino acid, and in these
cases the complex is usually disrupted and the tRNA, recycled.
16

The ribosome has three sites for tRNA to bind
E P A
Large- subunit
Small- subunit
The aminoacyl site (A-site)
binds the incoming tRNA
with the complementary
codon on the mRNA
The peptidyl site (P-
site) holds the tRNA
with the growing
polypeptide chain.
The exit (E-site) holds the tRNA
without its amino acid.
17

Interaction of Ribosome with mRNA and tRNA
 When an aminoacyl-tRNA initially binds to
its corresponding codon on the mRNA, it is
in the A site.
 Then, a peptide bond forms between the
amino acid of the tRNA in the A site and the
amino acid of the charged tRNA in the P
site.
 The growing polypeptide chain is
transferred to the tRNA in the A site.
 Translocation occurs, moving the tRNA in
the P site, now without an amino acid, to
the E site; the tRNA that was in the A site,
now charged with the polypeptide chain, is
moved to the P site.
 The tRNA in the E site leaves and another
aminoacyl-tRNA enters the A site to repeat
the process. 19

DNA v/s mRNA: Extra information
DNA
 Regulatory sequences: site for the binding
of regulatory proteins; the role of regulatory
proteins is to influence the rate of
transcription.
 Promoter: site for RNA polymerase binding;
signal the beginning of transcription.
 Terminator: signal the end of transcription.
mRNA
 Ribosomal binding site: site for ribosome
binding; translation begin near this site in
the mRNA. The ribosome scans the mRNA
for start codon.
 Start codon: Specify the first amino acid in
a polypeptide sequence, usually a
formylmethionine (in bacteria) or a
methionine (in eukaryotes).
 Codons: 3-nucleotide sequence within the
mRNA that specify particular amino acids
within a polypeptide.
 Stop codon: specifies the end of
polypeptide synthesis.
20

Translation
 Translation is the process in which ribosomes in the cytoplasm or
endoplasmic reticulum synthesize proteins from mRNA.
 During translation, an mRNA sequence is read using the genetic code,
which is a set of rules that defines how an mRNA sequence is to be
translated into the 20-letter code of amino acids, which are the building
blocks of proteins.
 The genetic code is a set of three-letter combinations of nucleotides called
codons, each of which corresponds with a specific amino acid or stop
signal.
 The polypeptide later folds into an active protein and performs its functions
in the cell.
 The ribosome facilitates decoding by inducing the binding of
complementary tRNA anticodon sequences to mRNA codons.
 The tRNAs carry specific amino acids that are chained together into a
polypeptide as the mRNA passes through and is "read" by the ribosome.
21

The Mechanism of Translation: Initiation
The initiation of protein synthesis begins with the formation of an initiation complex.
 In E. coli, this complex involves the small 30S ribosome, the mRNA template, initiation factors that help the
ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator
tRNA carrying N-formyl-methionine (fMet-tRNAfMet).
 The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet).
 Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain
synthesized by E. coli.
 In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also
known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA
molecules that compose the ribosome.
 This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the
50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.
In eukaryotes, initiation complex formation is similar, with the following differences:
 The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi
 Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5′
cap of the eukaryotic mRNA, then tracks along the mRNA in the 5′ to 3′ direction until the AUG start codon is
recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.
24

Shine–Dalgarno sequence
 The Shine–Dalgarno sequence is a ribosomal binding site in bacterial and
archaeal messenger RNA, generally located around 8 bases upstream of
the start codon AUG.
 The RNA sequence helps recruit the ribosome to the messenger RNA
(mRNA) to initiate protein synthesis by aligning the ribosome with the start
codon.
25

Is the Shine Dalgarno sequence present in eukaryotes?
 Eukaryotic mRNA does not have a Shine–Dalgarno sequence.
 Instead, eukaryotic ribosomes recognize the 5′ cap structure, and the
Kozak sequence, which is a loosely conserved sequence found around
the first AUG.
27

The Mechanism of Translation: Elongation
 In prokaryotes and eukaryotes, the basics of elongation of translation are the
same.
 In E. coli, the binding of the 50S ribosomal subunit to produce the intact ribosome
forms three functionally important ribosomal sites:
 The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P
(peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide
bonds with the growing polypeptide chain but have not yet dissociated from their
corresponding tRNA.
 The E (exit) site releases dissociated tRNAs so that they can be recharged with
free amino acids. There is one notable exception to this assembly line of tRNAs:
During initiation complex formation, bacterial fMet−tRNAfMet or eukaryotic Met-tRNAi
enters the P site directly without first entering the A site, providing a free A site
ready to accept the tRNA corresponding to the first codon after the AUG.
28

 Elongation proceeds with single-codon movements of the ribosome each called a
translocation event.
 During each translocation event, the charged tRNAs enter at the A site, then
shift to the P site, and then finally to the E site for removal.
 Ribosomal movements, or steps, are induced by conformational changes that
advance the ribosome by three bases in the 3′ direction.
 Peptide bonds form between the amino group of the amino acid attached to the A-
site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA.
 The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-
based ribozyme that is integrated into the 50S ribosomal subunit.
29

 The amino acid bound to the P-site tRNA is also linked to the growing polypeptide
chain.
 As the ribosome steps across the mRNA, the former P-site tRNA enters the E site,
detaches from the amino acid, and is expelled.
 Several of the steps during elongation, including binding of a charged aminoacyl
tRNA to the A site and translocation, require energy derived from GTP hydrolysis,
which is catalyzed by specific elongation factors.
 Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each
amino acid, meaning that a 200 amino-acid protein can be translated in just 10
seconds.
30

The Mechanism of Translation: Termination
 The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA)
is encountered for which there is no complementary tRNA.
 On aligning with the A site, these nonsense codons are recognized by release
factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching
from its tRNA, releasing the newly made polypeptide.
 The small and large ribosomal subunits dissociate from the mRNA and from each
other; they are recruited almost immediately into another translation initiation
complex.
31

Comparison of Translation in Bacteria versus Eukaryotes
Property Bacteria Eukaryotes
Ribosomes 70S
30S (small subunit) with 16S rRNA
subunit
50S (large subunit) with 5S and
23S rRNA subunits
80S
40S (small subunit) with 18S rRNA
subunit
60S (large subunit) with 5S, 5.8S, and
28S rRNA subunits
Amino acid carried by
initiator tRNA
fMet Met
Translation recognition
site
Shine-Dalgarno sequencein Cap or
Kozak sequence
Simultaneous
transcription and
translation
Yes No
32

Key concepts of translation
tRNAs bring amino acids to the ribosomes during translation to be
assembled into polypeptide chains.
 tRNAs are encoded by tRNA genes.
 All tRNA molecules are similar in size and shape.
 All tRNAs have CCA at the 3' end to which the amino acid attaches.
 At the other "end" of the tRNA molecule is the anticodon, which, during
translation, "reads" the matching codon on the mRNA.
33

An enzyme called aminoacyl-tRNA synthetase adds the correct amino
acid to its tRNA.
 The correct amino acid is added to its tRNA by a specific enzyme called an
aminoacyl-tRNA synthetase. The process is called aminoacylation, or
charging.
 Since there are 20 amino acids, there are 20 aminoacyl-tRNA synthetases.
 All tRNAs with the same amino acid are charged by the same enzyme,
even though the tRNA sequences, including anticodons, differ.
34

Translation initiation.
 An initiation complex for translation forms by the assembly of the ribosomal
subunits and initiator tRNA (met-tRNA) at the start codon on the mRNA.
35

Elongation of the Polypeptide Chain.
 Elongation of the polypeptide chain begins by the appropriate aminoacyl-
tRNA binding to the codon in the A site of the ribosome.
36

Termination of Translation.
 At a stop codon, a release factor reads the triplet, and polypeptide
synthesis ends; the polypeptide is released from the tRNA, the tRNA is
released from the ribosome, and the two ribosomal subunits separate from
the mRNA. Polypeptide synthesis repeats until a stop codon is reached.
37

Polysomes
 Several ribosomes can translate an mRNA at the same time, forming what
is called a polysome.
 More than one ribosome can translate an mRNA at one time, making it
possible to produce many polypeptides simultaneously from a single
mRNA.
38

Significance of translation
 Translation is very important in the process of making proteins.
 Without transcription and translation, our body would have no possible way to
make proteins, or function.
 Proteins allow our body to do everything.
Muscle proteins allow our muscles to strengthen and grow.
Antibodies protect the body from germs.
Some proteins support body structures, whereas others help with your body’s
movements.
There are thousands of functions for different proteins.
39

Post-translational
modifications
 Many proteins undergo chemical modifications at certain amino acid residues following translation.
 These modifications are essential for normal functioning of the protein and are carried out by one or more
enzyme catalyzed reactions.
 Post-translational modifications of proteins, which are not gene-template based, can regulate the protein
functions, by causing changes in protein activity, their cellular locations and dynamic interactions with other
proteins.
40

Post-translational
modifications
 Post translational modifications refer to any alteration in the amino acid sequence of the
protein after its synthesis.
 It may involve the modification of the amino acid side chain, terminal amino or carboxyl
group by means of covalent or enzymatic means following protein biosynthesis.
 Generally, these modifications influence the structure, stability, activity, cellular localization
or substrate specificity of the protein.
 Post translational modification provides complexity to proteome for diverse function with
limited number of genes.
41

Part 1: Overview of PTMs
PTMs Detection methods (Extra information)
Part 2: Gel-based detection techniques for PTMs
Part 3: MS-based detection techniques for PTMs
Part 4: Microarray-based detection techniques for PTMs
Phosphorylation
Glycosylation
Acylation
Alkylation
Hydroxylation
Commonly observed PTMs
Cytosol
Endoplasmic reticulum (ER)
P
P
Glc
Glc
CH3
CH3
Signal
sequence
Cleaved
protein
PTMs
Protein
translation
Post Translational Modification
42

Post Translation
Modifications: Detection
methods
Part 1: Overview and Importance
43

Part 1: Overview of Post Translational Modification
 Post-translational modification (PTM): 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. Some of the most commonly observed PTMs include:
 Phosphorylation: The process by which a phosphate group is attached to certain amino acid side chains in
the protein, most commonly serine, threonine and tyrosine.
 Glycosylation: Glycosylation is the addition of carbohydrate molecules to the polypeptide chain and
modifying it into glycoproteins. Many of the proteins that are destined to become a part of plasma membrane
or to be secreted from the cell, have carbohydrate chains attached to the amide nitrogen of
asparagine(N linked) or the hydroxyl groups of serine, threonine(O linked). N glycosylation occurs in
ER and O glycosylation occurs in the golgi complex.
 Sulfation: Sulfate modidication takes place by the addition of sulphate molecules and these modifications of
proteins occurs at tyrosine residues. Tyrosine sulfation accomplished via the activity of
tyrosylproteinsulfotransferases (TPST) which are membrane associated enzymes of trans-Golgi network.
There are two known TPSTs. TPST-1 TPST-2 The universal phosphate donor is 3’-phosphoadenosyl- 5’-
phosphosulphate (PSPA).
Types
44

 Alkylation/ Methylation: Addition of alkyl groups, most commonly a methyl group to amino acids such as
lysine or arginine. Other longer chain alkyl groups may also be attached in some cases. The transfer of one-
carbon methyl groups to nitrogen or oxygen to amino acid side chains increases the hydrophobicity of the
protein and can neutralize a negative amino acid charge when bound to carboxylic acids. Methylation is
mediated by methyltransferases and S-adenosyl methionine (SAM) is the primary methyl group donor.
 Hydroxylation: This PTM is most often found on proline and lysine residues which make up the collagen
tissue. It enables crosslinking and therefore strengthening of the muscle fibres. The biological process of
addition of a hydroxy 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 the 20 amino acids,
the two amino acids regulated by this method are proline and lysine.
 SUMOylation: SUMO (small ubiquitin related modifier) proteins are 100 amino acid residue proteins which
bind to the target protein in the same way as ubiquitin. They also confer the transcription regulatory activity of
the protein and help in the transport of the target protein from cytosol to the nucleus.
 Acylation: The process by which an acyl group is linked to the side chain of amino acids like aspargine,
glutamine or lysine.
Types
45

 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.
 Cytosol: A cellular compartment that serves as the site for protein synthesis.
 Signal sequence: A sequence that helps in directing the newly synthesized polypeptide chain to its
appropriate intracellular organelle. This sequence is most often cleaved following protein folding
and PTM.
 Endoplasmic reticulum: A membrane-bound cellular organelle that acts as a site for post-
translational modification of the newly synthesized polypeptide chains.
 Cleaved protein: The protein product obtained after removal of certain amino acid sequences such
as N- or C-terminal sequences, signal sequence etc.
Key terminologies
46

Cytosol
Endoplasmic
reticulum
(ER)
P
P
Glc
Glc
CH3
CH3
Cleaved
protein
Protein folding
& PTMs
mRNA
Ribosome
Protease
Removal of
certain N- and
C-terminal
residues
 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.
Translated
Protein
Process of post-translational modification
47

Phosphorylation
Glycosylation
Acylation
Alkylation
Hydroxylation
Ser, Thr, Tyr
Asn, Ser, Thr
Asn, Gln, Lys
Lys, Arg
Pro, Lys
 There are several types of post translational modifications that can take place at different amino acid residues.
 The most commonly observed PTMs include phosphorylation, glycosylation, methylation as well as hydroxylation and
acylation.
 Many of these modifications, particularly phosphorylation, serve as regulatory mechanisms for protein action.
Different types of PTMs & their modification sites
48

A C G G U G C C G U G C A C G
A C A C U A C G C A C U
Gene sequence
Expected protein
structure
Actual protein
structure
P
CH3
Glc
 The final structure of functional proteins most often does not correlate directly with the corresponding gene sequence.
 This is due to the PTMs that occur at various amino acid residues in the protein, which cause changes in interactions between
the amino acid side chains thereby modifying the protein structure.
 This further increases the complexity of the proteome as compared to the genome.
Increased complexity of proteome due to PTMs
49

Ser
R
CH2
CH
CH3
CH2
Thr
Tyr
ATP ADP
Kinase
Amino acid
residue
Phosphorylated
residue
OH
C
NH3
+
COO-
R
H O
C
NH3
+
COO-
R PO4
3-
H
 Phosphorylation of amino acid residues is carried out by a class of enzymes known as kinases that most commonly modify side
chains of amino acids containing a hydroxyl group.
 Phosphorylation requires the presence of a phosphate donor molecule such as ATP, GTP or other phoshorylated substrates.
 Serine is the most commonly phosphorylated residue followed by threonine and tyrosine.
 Removal of phosphate groups is carried out by the phosphatase enzyme and thus this forms one of the most important
mechanisms for regulation of proteins.
Phosphorylation reaction
50

Ser/Thr
Asn
Glycosyl transferase
N-linked Glycosylation
O-linked Glycosylation
Glycosyl transferase
Sugar residues
N-linked amino acid
O-linked amino acid
 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, N-acetylglucosamine, N-
acetylgalactosamie as well as fucose.
CONH2
C
NH3
+
COO-
CH2
H
CON
C
NH3
+
COO-
CH2
H
OH
C
NH3
+
COO-
R
H
O
C
NH3
+
COO-
R
H
Glycosylation reactions
51

PTMs have significant biological functions which include:
 Aids in proper protein folding – few lectin molecules called calnexin binds to glycosylated proteins and assist
in its folding.
 Confers stability to the protein- glycosylation can modify the stability of the protein by increasing protein half
life.
 It protects the protein against cleavage by proteolytic enzyme by blocking the cleavage sites.
 Protein sorting or translocation- If phosphorylated mannose residues are present in the protein it always goes
to lysosome.
 It regulates protein activity and function- phosphorylation of protein is a reversible PTM which activates the
protein.
 Acetylation regulates many diverse functions, including DNA recognition, protein-protein interaction and
protein stability.
 Redox-dependent PTM of proteins is emerging as a key signaling system conserved through evolution,
influences many aspects of cellular homeostasis.
 PTMs are important components in cell signaling, as for example when prohormones are converted
to hormones.
 It significantly increases the diversity and complexity in the proteome. 52

Post Translation
Modifications:
Detection methods
(Extra information)
53

1. Pro-Q-diamond
2. Immunoblotting
Gel staining
Gel scanning
SDS-PAGE/
2-DE gel
Nitrocellulose sheet
Blotting
Electrophoresis
Specific probe
antibodies
Detection
Labeled
secondary
Abs
54

1. Pro-Q-diamond: This fluorescent dye is capable of detecting modified proteins that have been phosphorylated at their
serine, threonine or tyrosine residues. They are suitable for use with electrophoretic techniques or with protein microarrays
and offer sensitivity down to few ng levels, depending upon the format in which they are used. This dye can also be
combined with other staining procedures thereby allowing more than one detection protocol on a single gel.
a) Gel staining: The process by which the protein bands on an electrophoresis gel are stained by suitable dyes for
visualization.
b) Gel scanning: The visualization of the stained protein bands on an electrophoresis gel by exciting it at a suitable
maximum wavelength such that the dye absorbs the light and emits its own characteristic light at another emission
wavelength.
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.
a) Electrophoresis: Electrophoresis is a gel-based analytical technique that is used for separation and visualization of
biomolecules like DNA, RNA and proteins based on their fragment lengths or charge-to-mass ratios using an electric field.
The protein mixture is first separated by means of a suitable electrophoresis technique such as SDS-PAGE or Two-
dimensional Electrophoresis.
Definitions of the components:
55

b) Blotting: The process by which the proteins separated on the electrophoresis gel are transferred on to another surface such as
nitrocellulose by placing them in contact with each other.
c) Nitrocellulose sheet: A membrane or sheet made of nitrocellulose onto which the protein bands separated by electrophoresis
are transferred for further probing and analysis.
d) Specific probe antibodies: Antibodies that are specific to a particular protein modification can be used as probes to detect
those proteins containing that particular PTM. Protein phosphorylation is commonly detected using anti-phosphoserine,
phosphothreonine or phosphotyrosine antibodies. Recently, specific motif antibodies have also been developed which detect a
particular sequence of motif of the protein that contains a PTM.
e) Labeled secondary Abs: Antibodies labeled with a suitable fluorescent dye molecule are used to detect the interaction
between the modified protein and its antibody by binding to another domain of the probe antibody.
56

Pro-Q-diamond staining
Completed 2-DE gel
Protein bands get
fixed on gel and
minimize diffusion.
Tubing connected &
outlet opened
Dye stains the
phosphorylated
protein bands
only.
Excess dye
removed
 Protein phosphorylation can be detected using a novel gel-based detection technique.
 Proteins separated on a 2-DE gel are first placed in a fixing solution containing methanol and acetic acid which fixes the protein
bands on to the gel and minimizes any diffusion.
 They are then stained using the Pro-Q-diamond staining solution which selectively stains only phosphoproteins on the gel.
 The excess stain is then washed off with a solution of methanol and acetic acid.
Tray with fixing
solution (methanol +
acetic acid)
Pro-Q-diamond stain
Washing solution (methanol + acetic
acid)
57

Decreasing
molecular
weight
Decreasing pH
Gel scanner
Emission maxima
– 580 nm
 The stained gel is then scanned at its excitation wavelength using a gel scanner.
 The gel image obtained shows the protein bands corresponding to only the phosphoproteins present.
 This image is saved and the gel is then removed from the scanner for treatment with the second stain, a procedure known as
dual staining.
Phosphoprotein
image
Stained gel
Gel removed
from scanner
Gel scanning
58

 The scanned gel is then removed from the scanner and placed in the SYPRO-Ruby Red fluorescent dye solution.
 This dye stains all the protein spots present on the gel thereby providing a total protein image with sensitivity down to
nanogram level.
 Excess dye is then washed off using a solution of methanol and acetic acid.
SYPRO-Ruby red
staining solution
Tubing connected &
outlet opened
Dye stains all
protein bands.
Excess dye
removed
Washing solution
(methanol + acetic
acid)
Dual staining with SYPRO-Ruby Red
59

Decreasing
molecular
weight
Decreasing pH
Gel scanner
 The gel stained with SYPRO-Ruby Red is then scanned in the gel scanner at its excitation maxima.
 The image produced will have more number of spots since all proteins present on the gel are detected.
 This dual staining procedure provides a useful comparative profile of the phosphoproteins and the total proteins on the gel,
thereby enabling detection of the phosphorylated proteins.
Emission maxima
– 610 nm
Total protein
image
Fluorescence
Phosphoprotein
image
Fluorescence
Total protein image by
SYPRO-Ruby Red
A comparative profile
between total protein image
and phosphoprotein image
enables detection of
phosphorylated proteins.
Phosphoprotein
image
Stained gel
Gel scanning
60

Direction of
migration
Anod
e
Cathode
-
+
Buffer
Acrylamide
gel
Sample loading
Protein
mixture
SDS-PAGE 2-D
Electrophoresis
Proteins focused on
IPG strip
Direction of
migration
Completed
stained gels
 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.
 These separated proteins on the gel are used for further analysis.
Immunoblotting step -1
61

Immunoblotting
Completed
gels
Nitrocellulose
sheet
Blotting
Specific phospho-
tyrosine
antibodies added
Detection using
labeled secondary
antibodies
 The separated protein bands are then blotted onto a nitrocellulose membrane.
 These membranes are then probed either by means of specific anti-phospho-amino acid antibodies or more recently, by motif
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.
 Thus, the use of immunoblotting technique has been shown to be extremely effective for detection of PTMs.
Proteins
phosphorylated at
Tyr residues
Proteins
phosphorylated at
Tyr residues
Immunoblotting step-2
62

1. MALDI-TOF analysis
MALDI-TOF-MS
Ion source
Flight tube
Detector
2. LC-MS/MS approach
Liquid
chromatography
Tandem MS
Affinity columns
63

1. 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.
It has the following components:
a) Ion source: The ion or ionization source is responsible for converting analyte molecules into gas phase ions in vacuum. The
technology that enables this is termed soft ionization for its ability to ionize non-volatile biomolecules while ensuring minimal
fragmentation and thus, easier interpretation. In MALDI-TOF-MS, the ion source used is MALDI, in which the target analyte is
embedded in dried matrix-sample and exposed to short, intense pulses from a UV laser.
b) Flight tube: Connecting tube between the ion source and detector within which the ions of different size and charge migrate to
reach the detector. 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.
c) Detector: The ion detector determines the mass of ions that are resolved by the mass analyzer and generates data which is
then analyzed. The electron multiplier is the most commonly used detection technique.
Definitions of the components
64

2. LC-MS/MS approach: LC-MS/MS a common analytical tool that combines physical separation by liquid chomatography with
mass analysis and resolution by mass spectrometry. It is capable of separating and identifying complex mixtures for proteomics
studies.
a) Liquid chromatography: This is a chromatographic separation technique that separates molecules based on their differential
adsorption and desorption between the stationary matrix phase in the column and the mobile phase.
b) Affinity columns: Columns that make use of specific affinity interactions between the analyte of interest and the bound
stationary phase matrix thereby successfully separating this component from a complex mixture. Immobilized Metal ion Affinity
Chromatography (IMAC) is one such affinity technique that relies on the formation of specific coordinate-covalent bonds between
certain amino acid residues of the protein (like histidine) and the immobilized metal ions. Phosphorylated proteins have been
found to bind specifically to ions such as iron, gallium and zinc, thus facilitating their separation by IMAC. Recently, titanium
dioxide (TiO2) columns have proved to be extremely useful for specific separation of phosphorylated proteins.
c) Tandem MS: This is a mass spectrometry technique that makes use of a combination of ion source and two mass analyzers,
separated by a collision cell, in order to provide improved resolution of the fragment ions. The mass analyzers may either be the
same or different. The first mass analyzer usually operates in a scanning mode in order to select only a particular ion which is
further fragmented and resolved in the second analyzer. This can be used for protein sequencing studies.
Definitions of the components
65

Trypsin
digestion
PTM modified
protein of
interest
Digested
protein
Protein
Matrix
196 –well MALDI Plate
 Post translational modifications can be detected by means of mass spectrometry due to the unique fragmentation patterns of
phosphorylated seine and threonine residues..
 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.
MALDI-TOF analysis – Digestion & sample spotting
66

Matrix & analyte
Target plate
Laser
Detector
Flight tube
MALDI
 The target plate containing the spotted matrix and analyte is placed in a vacuum chamber with high voltage and short laser
pulses are applied.
 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.
MALDI-TOF analysis – Ionization & detection
67

 Identification of PTMs by MS largely lies in the interpretation of results.
 Comparison of the list of observed peptide masses from the spectrum generated with the expected peptide masses enables
identification of those peptide fragments that contain any PTM due to the added mass of a modifying group. In this hypothetical
example, two peptide fragments are found to have different m/z values, differing by 80 daltons and 160 daltons.
 It is known that the added mass of a phosphate group causes an increase in m/z of 80 daltons. Therefore, this principle of
mass difference enables detection of modified fragments.
m/z
Relative
abundance
25
32
53
65
72
81
92
107
122
143
151
164
170
m/z
Relative
abundance
25
192
53
65
152
81
92
107
12
2
143
151
16
4
170
m/z
Relative
abundance
25
32
53
65
72
81
92
107
122
143
151
164
170
15
2 192
80 Da
160 Da
Expected peptide
masses
Observed peptide
masses
Superimposed image
80 Da implies
presence of 1
phosphate group!
Presence of 2
phosphate group!
Thus, examination and comparison of list of observed
peptide masses with expected peptide masses enables
simple detection of PTMs.
MALDI-TOF analysis – Data interpretation
68

 Liquid chromatography coupled with mass spectrometry serves as a useful technique for enrichment and identification of proteins
having a particular type of PTM from a complex mixture.
 The complex protein sample is loaded onto a miniaturized affinity column which will interact specifically with proteins having the
PTM of interest.
 Here, we depict the use of immobilized metal affinity chromatography columns containing ions such as Ga3+, Zn2+, Fe3+ or TiO2
which have been found to specifically chelate the phosphorylated proteins.
 Unwanted proteins are removed by washing the column with a suitable buffer solution after which the phosphorylated protein of
interest is eluted out by modifying the buffer solution.
Sample protein
mixture
Miniaturized
immobilized metal
affinity columns
Direction of
migration
PO4
3-
PO4
3-
M3+
M3+
M3+
PO4
3-
Metal ions
Phosphorylated
residue of
protein
IMAC: Ga3+, Zn2+, Fe3+
Other affinity columns: TiO2
Buffer
solution
Phosphorylated
protein remains
bound
Buffer
solution 2
Purified phosphorylated
protein
TiO2 columns were found to have
better selectivity and sensitivity of
detection for phosphorylated peptide
binding when compared to IMAC.
LC-MS/MS based approach – Liquid chromatography
69

 The protein purified by liquid chromatography is then subjected to typtic digestion followed by analysis using tandem mass
spectrometry.
 Here we demonstrate the use of MALDI-TOF-TOF-MS for resolution of the generated ion fragments.
 Separation is based on the flight time of the ions and greater resolution is achieved due to the presence of two mass analyzers.
 The peptide ion spectrum generated is analyzed by comparing it with the expected spectrum, thereby allowing determination of
modified peptides having different m/z values.
Reflector
TOF 2
(RF
mode)
TOF 1
(scanning
mode)
Collision cell
Detector
Purified protein Digested
protein
MS/MS
analysis
Analysis and
interpretation of data
is carried out as
described for MALDI-
TOF-MS.
LASER
LC-MS/MS based approach – Tandem mass spectroscopy
The number of electrons
removed is the charge number
(for positive ions). m/z
represents mass divided by
charge number and the
horizontal axis in a mass
spectrum is expressed in units
of m/z. Since z is almost
always 1 with GCMS, the m/z
value is often considered to be
the mass.
70

1. Protein microarrays
2. Antibody microarrays
[g-33P]
ATP
ADP
Kinase
enzyme
Protein array
Phosphorylated
proteins
Autoradiography film
`
Antibody microarrays
Labeled protein
mixture
Phosphorylated
proteins
Array
scanning
71

1. Protein microarrays: These are miniaturized arrays normally made of glass, onto which small quantities of many proteins can be
simultaneously immobilized and analyzed. For detection of phosphorylation sites, potential protein substrates are immobilized on to the array.
a) Kinase enzyme: An enzyme that is responsible for phosphorylation of specific amino acid residues in the protein with the help of ATP as a
phosphate donor.
b) Phosphorylated proteins: Proteins that have been phosphorylated at specific amino acid residues.
c) Autoradiography: Radioactivity is the process by which certain elements spontaneously emit energy in the form of particles or waves due
to disintegration of the unstable atomic nuclei into a more stable form. These radiations that are given out can be detected by means of
autoradiography, wherein the radiations are allowed to strike a photographic film which on exposure shows the presence radioactive
emissions.
2. Antibody microarrays: An array onto which different antibodies are spotted, which have specific binding domains for detection of the
protein of interest from a complex mixture. For detection of PTMs, antibodies against specific protein motifs containing the PTM or against a
specific residue containing a phosphorylated site may be used.
a) Labeled protein mixture: The protein mixture containing the protein of interest is labeled uniformly with a suitable fluorescent dye which
can be detected by scanning at the appropriate wavelength. Cyanine dyes are commonly used for such labeling purposes.
b) Array scanning: Once the binding interactions have taken place on the array surface and excess unbound material has been washed
away, the array is scanned using a microarray scanner. This scans the array at a suitable wavelength depending upon the fluorescent dye
used for labeling purposes to generate an image depicting the array positions at which binding has occurred.
72

 PTMs can also be detected by means of protein microarrays using a kinase assay.
 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.
[g-33P]
ATP
ADP
Proteome array containing
potential substrates for
phosphorylation
Kinase
enzyme
[g-33P] ATP
solution
Ser Ser
Protein
substrate
Phosphorylated
protein
Kinase enzyme
Protein microarrays
73

 After sufficient incubation, excess unbound ATP and enzyme are washed off the array surface.
 Detection is carried out by means of autoradiography wherein a photographic film is placed in contact with the array surface.
 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.
 Thus proteome chip technology offers a useful platform for detection of phosphporylated proteins.
Proteome array
Washing
Phosphorylated
proteins
Detection-
Autoradiography
film
33P
33P
33P
33P
33P
Developed image
Radioactive
emissions
Protein microarrays
74

 Antibodies specific to phosphorylated serine, threonine or tyrosine residues as well as motif antibodies can be immobilized on
to a suitably coated microarray surface and used for detection of PTM.
 The complex protein mixture containing modified and unmodified proteins is labeled with a suitable fluorescent tag molecule
and added to the array surface.
 Specific binding interactions occur between the phosphorylated proteins and their corresponding antibodies.
Polymer coated
glass slide
Anti-phospho serine, threonine and
tyrosine antibodies immobilized on
array
Labeled protein
mixture added
Specific binding of
phosphorylated
proteins
75

 The array is then washed to remove any excess unbound proteins from the surface.
 This is followed by scanning of the array using a microarray scanner at a suitable wavelength to detect the fluorescent tag of
the bound proteins.
 This method offers sensitive and simultaneous detection of large number of post translationally modified proteins.
Bound phosphorylated
proteins
Array washing
Unbound proteins
removed
Microarray scanner
Array image
76

1. Proline most commonly undergoes which of the following PTMs?
Answers: a) Glycosylation b) Phosphorylation c) Hydroxylation d) Acylation
2. N-linked glycosylation most commonly occurs on which of the following amino acids?
Answers: a) Serine b) Threonine c) Aspartic acid d) Aspargine
3. Which of the following fluorescent dyes is used for specific detection of phosphoproteins?
Answers: a) Pro-Q-diamond
b) SYPRO-Ruby Red
c) SYPRO-Ruby Orange
d) Coomassie brilliant blue
4.Difference in molecular weight of 240 Da between two peptide fragments obtained by MALDI-TOF-MS is indicative of presence
of how many phosphorylation sites on the protein?
Answers: a) 2 b) 3 c) 0 d) 5
5. Which of the following metal ions is not used for separation of phosphorylated proteins during liquid chromatography?
Answers: a) Zn2+ b) Fe3+ c) Ga3+ d) K+
Questions
77

Translation: Protein synthesis

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Translation: Protein synthesis