College of Natural and Computational Science

Department of Biotechnology

Post Graduate Program (Msc. In Plant Biotechnology)

Plant Molecular Biology

Title: Post Translation Modification.

Prepared By: Samson Zemikael

 Introduction
 Definition
 Types of Post Translation Modification
 Methods to Detect Protein Posttranslational Modifications
 Summary
 The proteins synthesized in translation are, as such, not functional. Many
changes take place in the polypeptides after the initiation of their synthesis.
 most frequently, after the protein synthesis is completed. These
modifications include protein folding, trimming by proteolytic degradation,
splicing and covalent changes which are collectively known as
modifications.
 Post-translational modifications refer to any change in the chemical
composition of proteins following translation.
 This modification increase the functional diversity of the proteome by the
covalent addition of functional groups (https//www.slideshare.net).
 Many proteins undergo chemical modifications at certain amino acid
residues following translation.
 These chemical modifications are necessary for the transport of proteins
across the plasma membrane.
 2.1.lntein splicing
o Inteins are intervening sequences in certain proteins. These are comparable
to introns in mRNAs. lnteins have to be removed, and exteins ligated in the
appropriate order for the protein to become active.

2.2. Proteolytic degradation

o Many proteins are synthesized as the precursors which are much bigger in
size than the functional proteins. Some portions of precursor molecules are
removed by proteolysis to liberate active proteins. This process is
commonly referred to as trimming. The formation of insulin from
preproinsulin, conversion of zymogens (inactive digestive enzymes e.g.
trypsinogen) to the active enzymes are some examples of trimming.
 The addition of ubiquitin to a substrate protein is called ubiquitination.
 Ubiquitin is a small regulatory protein found in most tissues of eukaryotic
organisms and was discovered in 1975.
 This affects proteins in many ways:
 it can mark them for degradation via the proteasome,
 alter their cellular location, affect their activity, and promote or
 prevent protein interactions.
 Ubiquitination requires 3 enzymes:
 E1 (ubiquitin-activating enzyme) activates ubiquitin (U)
 E2 (ubiquitin-conjugating enzyme) acquires U via high-energy
 E3 (ubiquitin ligase) transfers U to target proteins
 Hierarchical organization: one or few E1s exist, more E2s, many E3s.
 The proteins synthesized in translation are subjected to many covalent
changes. By these modifications in the amino acids, the proteins may be
converted to active form or inactive form.

2.3.1. Phosphorylation
o Phosphorylation: The addition of phosphate group covalently to the amino
acid for modification. The hydroxyl group containing amino acids of
proteins, namely serine, threonine and tyrosine are subjected to
phosphorylation. The phosphorylation may either increase or decrease the
activity of the proteins.

o Group of enzymes called protein kinases catalyse phosphorylation.

 Phosphorylation requires the presence of a phosphate donor molecule such
as ATP, GTP or other phoshorylated substrates.
  Acetylation is transfer of an acetyl group to a nitrogen molecule through the

addition of an acetyl group from acetyl-CoA by N-acetyl transferase (NAT)

(E.Seto,2017).

 Purpose:

 Put the target protein in its exact place

,
 Glycosylation is the process by which a carbohydrate is covalently attached
to a target macromolecule, typically proteins and lipids ( V. Nairn ,2012).
 This modification serves various functions:
o For instance, some proteins do not fold correctly unless they are
glycosylated.
o In other cases, proteins are not stable unless they contain oligosaccharides
linked at the amide nitrogen of certain asparagine.
 Also plays a role in cell-cell adhesion (a mechanism employed by cells of
the immune system) via sugar-binding proteins called lectins, which
recognize specific carbohydrate moieties.
 In addition, glycosylation is often used by viruses to shield the underlying
viral protein from immune recognition
 This major type of PTM has significant applications for protein folding,
conformation, distribution, stability, and activity.

COO-

H C CH2 O

NH3+
 The transfer of one-carbon methyl groups to amino acid side chains to

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.

 At the molecular level, the addition of a methyl moiety to protein serves as

a signal to directly regulate modular protein-protein interactions.


 The reaction of proteins with a variety of free radicals and reactive oxygen
species (ROS) leads to oxidative protein modifications such as formation
protein
 hydro peroxides,
 hydroxylation of aromatic groups and aliphatic amino acid side chains
 oxidation of sulfhydryl groups, oxidation of methionine residues,
conversion of some amino acid residues into
 carbonyl groups,
 cleavage of the polypeptide chain, and
 formation of cross-linking bonds.
 Aromatic and sulfur-containing residues are particularly susceptible to
oxidative modification (P. Campbel,2006).
 Nitrosylation is addition of a nitric oxide (NO) to cysteine residues, forming
nitrosothiols (NOs), via redox-mediated reactions.

 Is method by which nitric oxide exerts it effects on cells is by the process of S-

nitrosylation. In this signaling mechanism, nitric oxide modifies the sulfur atom of
a protein cysteine residue, forming an S-nitrosothiol group. This process requires
no enzyme, and many S-nitrosylated proteins have altered function.

 Nitrosylation is used by cells to stabilize proteins, regulate gene expression, and

provide NO donors. NO generation, localization, activation, and catabolism are
tightly regulated, and

 Nitrosylation reactions depend on catalytic amounts of transition metals, O2, O2−,

and pH.
 The detection of PTMs was carried out by various analytical
methods, such as:
Liquid chromatography, thin-layer chromatography, column
chromatography, and/or polyacrylamide gel electrophoresis (T.
Hunter,2001)
 Liquid chromatography serves as a useful technique for
enrichment and identification of proteins having a particular
type of PTM from a complex mixture.
 Here, we depict the use of immobilized metal affinity chromatography
columns containing ions such as Ga3+, Zn2+, Fe3+ which have been found
to specifically chelate the Modified (phosphorylated)proteins.
 Unwanted proteins are removed by washing the column with a suitable
buffer solution
 Then the phosphorylated protein of interest is eluted out by modifying the
buffer solution.
 Buffer solution
Miniaturized
immobilized metal
affinity columns

Phosphorylated protein remains

bound

Sample protein mixture

 First show the ‘protein mixture’ along with the ‘column’.
 This mixture should be poured into the column after which the ‘buffer
solution’ must be placed on the column.
 Liquid must flow into the column in the direction indicated.
 The column must be zoomed into to show the inset image on the right
where the green and violet circles must move towards each other, along
with the text below.
 Next, the pink and brown shapes must move from the column into the tube
below after which the ‘buffer solution’ must be replaced (change color).
 Finally the green figure must also move down from the column into the
tube below.
 PTMs play an important in :
 Modifying the end product of expression, contribute to
biological processes.
 playing a key role in many cellular processes such as cellular
differentiation, protein degradation, signaling and regulatory
processes, regulation of gene expression, and protein-protein
interaction.
 Post-translational modification (PTM) is refers to enzymatic alteration of
amino acid sequence of the protein. These modification detected by Liquid
chromatography method. Detection of the specific site of PTM can be achieved
by subsequent purification ( N. L. Kelleher, 2005). These modification
important for:
 Cellular differentiation,
 protein degradation, in proper protein folding , proper functioning
 Confers stability of protein,
 protects the protein against cleavage, regulates protein activity and protein-
protein interaction.
THANK YOU FOR YOUR ATTENTION!!!