Protein Engineering

BIT317 Class 2438

Lecture 10
Post-translational Modifications (PTM)
&
Chemical Modifications
(Modificomics)

The Well-Known Case

Post translational modifications are the key mechanism for increasing

proteomic diversity

It is not necessary that all proteins should under PTM

Some facts on Post translational modification

Proteome is dynamic and changes in response to stimuli, and post-translational
modifications are commonly employed to regulate cellular activity.
PTMs occur at distinct amino acid side chains or peptide linkages and are most
often mediated by enzymatic activity.
It is estimated that 5% of the proteome comprises enzymes that perform more than
200 types of post-translational modifications.
These enzymes include kinases, phosphatases, transferases and ligases, which
add or remove functional groups, proteins, lipids or sugars to or from amino acid
side chains, and proteases, which cleave peptide bonds to remove specific
sequences or regulatory subunits.
Many proteins can also modify themselves using autocatalytic domains, such as
autokinase and autoprotolytic domains.
Post-translational modification can occur at any step in the "life cycle" of a protein.
Protein PTMs can also be reversible depending on the nature of the modification.

Protein Phosphorylation / dephosphorylation

Reversibility

It is estimated that one-third of the proteins in the human proteome are

substrates for phosphorylation at some point (kinomics)
Occurs in Ser, Thr and Tyr

pS:pT:pY in a cell is 1800:200:1

Linear or Cascade?

Phosphorylation plays critical roles in the regulation of many cellular processes

including cell cycle, growth, apoptosis and signal transduction pathways.

Glycosylation
Protein glycosylation is acknowledged as one of the major post-translational
modifications, with significant effects on protein folding, conformation,
distribution, stability and activity.
Glycosylation encompasses a diverse selection of sugar-moiety additions to
proteins that ranges from simple monosaccharide modifications of nuclear
transcription factors to highly complex branched polysaccharide changes of cell
surface receptors.
Carbohydrates in the form of aspargine-linked (N-linked) or serine/threoninelinked (O-linked) oligosaccharides are major structural components of many cell
surface and secreted proteins.
In the ER, glycosylation is used to monitor the status of protein folding, acting
as a quality control mechanism to ensure that only properly folded proteins
are trafficked to the Golgi.

Glycosylation
Sugar moieties on soluble proteins can be bound by specific receptors in the
trans Golgi network to facilitate their delivery to the correct destination.
These sugars can also act as ligands for receptors on the cell surface to
mediate cell attachment or stimulate signal transduction pathways.
They can be very large and bulky, oligosaccharides can affect protein-protein
interactions by either facilitating or preventing proteins from binding to
cognate interaction domains. Because they are hydrophilic, they can also
alter the solubility of a protein.
Glycosylation increases the diversity of the proteome to a level unmatched
by any other post-translational modification. The cell is able to facilitate this
diversity, because almost every aspect of glycosylation can be modified,
including:
Glycosylation is thought to be the most complex post-translational
modification because of the large number of enzymatic steps involved

Proteolytic Processing
Cleavage of signal peptides
Translocation process
Digestive enzymes as inactive precursors (Zymogens)
Peptide harmones as preproproteins (eg. Insulin)
Protein splicing
Rarely seen (eg. Concanavalin a plant lectin)

Alteration of the chain termini

N-ter Met
Deformylase: removal of formyl group in prokaryotes
Met-aminopeptidase: removal of ter Met (easier if the second aa is
small) occurs in both prokaryotes and eukaryotes
Addition of ter-residues
Very rare assisted by tRNA very specific eg. Arginyl tRNAProtein transferase catalyses transfer of Arg to peptide with N-ter Glu / Asp
Acetylation of N-terminus
59 90% of eukaryotic proteins in cytoplasm
N-acetyltransferase acetyl CoA
Reaction depends on the second amino acid
The order could be [1] acetylation removal of N-ter
[2] removal of N-ter acetylation
Formation of pyroglutamyl residue if Gln is present self capping
Myristoylation
transferred from myristoyl CoA by N-myristoyl transferase
Occurs if Gly is the second residue and Ala/Ser/Asn/Gln/Val is the 3rd / 4th
Might aid in membrane attachment protein kinases / phosphotases

Lipid attachment
Myristoyl to N-ter
Glycosyl-phosphotidylinositol / farnesyl groups at the C-ter
Palmitoyl groups to the side chain of Cys

Sulfation
Tyr at Golgi tyrosyl protein sulfotransferase
Sulfate donor: 3-phosphoadenosine-5-phosphosulfate
Occurs in sequences rich in acidic residues

-carboxy-Glu residues
Monocarboxyl to dicarboxyl
Vitamin K dependent carboxylase
Bicarbonate as the carboxyl donor

Hydroxylation
Pro and Lys
Requires Oxygen, a-ketoglutarate and ascorbic acid
Catalyzed by prolyl 4-hydroxylase, lysyl 5-hydroxylase, prolyl 3-hydroxylase

Other modifications such as disulfide formation

Ubiquitation
Ribosylation
SUMOylation
ISGylation

as self reading topics

Overview of the amino acids susceptible for chemical reactions