basis of protein synthesis?
When proteins are synthesized, they consist of primary structure,
which is a sequence of amino acid residues joined covalently by
peptide bonds. They may also have disulfide bonds, forming
cystine from two cysteines, and these are also part of the primary
structure.
- As you know, the proteins can fold into secondary and tertiary
structure, and separate polypeptides can associate to form
proteins with 2 or more subunits (this is quaternary structure).
post-translational modifications?
Proteins (specific residues) can also undergo covalent modifications (R group modifications make the m molecule behave differently - imparting different properties) that allow them to perform different functions. Here we are going to examine these. These modifications occur separately from protein synthesis at translation, so they are called post-translational modification.
What does the phosphorylation process involve?
Phosphorylation of proteins involves the transfer of PO43- from adenosine triphosphate (ATP) to amino acid residues with hydroxyl groups, These
include Ser, Thr and Tyr. The phosphorylation of these three amino acids forms phosphoserine, phosphothreonine and phosphotyrosine.
What are protein kinases?
The addition of phosphate is catalyzed by enzymes called protein kinases.
What do protein phosphatases do?
The reverse reaction (i.e., the removal of phosphate to yield the original amino acid residue) is catalyzed by enzymes called protein phosphatases. This process reinstates the hydroxyl group.
What kind of proteins are typically phosphorylated?
Proteins that are phosphorylated often have signalling roles in the cell. A signal from outside of the cell can result in a change in the phosphorylation state of a protein inside the cell. This protein then carries out a role in response to
its phosphorylation state.
- There can also be phosphorylation cascades - which amplify the response.
Phosphorylation of tyrosine?
The phosphorylation of tyrosine involves the transfer of a phosphate (red) from ATP to the hydroxyl group on the tyrosine residue in a protein, giving phosphotyrosine and adenosine diphosphate (ADP). The reaction proceeds in the same way for serine and threonine.
Effects of phosphorylation on the residues?
The size and the charge of the molecule change when phosphorylation takes place.
- The phosphorylation of residues changes their sizes and imparts
two negative charges (on the phosphate group)
What is glycosylation?
Glycosylation of proteins involves the
transfer of carbohydrate to specific amino acid residues on a protein.
Where does glycosylation occur?
This most often occurs for extracellular
proteins, such as those in the blood plasma
or in the extracellular matrix.
What does glycoslyation change?
The carbohydrate that is covalently attached to the protein can change the behaviour of the protein, like: solubility of the protein, the half-life of the protein, the binding functions of the protein and the overall charge of the protein (when charged sugars are part of the carbohydrate).
How are carbohydrates added?
Carbohydrates are added by enzymes, primarily in the endoplasmic reticulum of the cell.
- they becoem attached during protein processing if it is synthesized and trafficked outside the cell - synthesized in rough ER and proteins with substrates are recognized by enzymes.
how does N-linked glycoslyation work?
In N-linked glycosylation, the
carbohydrate is covalently linked to
the nitrogen at the end of an Asn
residue in a protein.
* The bond between the amino acid
and the protein is called a
glycosydic bond.
* This occurs at particular amino acid
residue sequences in proteins: the
primary structure has to be Asn-X-
Thr/Ser, where X can be any amino
acid.
* The N-linked carbohydrates are
complex (many sugars linked
together) and some of the sugars
can be negatively charged
O-linked glycoslyation works how?
In O-linked glycosylation,
the carbohydrate is
covalently linked to the
hydroxyl group of Ser or
Thr. This bond is also
called a glycosidic bond.
- O-linked carbohydrates have only two sugars, as shown. They are never
complex. - Not every occurrence of Ser or Thr in a protein results in glycosylation. In fact, O-glycosylation is much less frequent than N-glycosylation.
- O-glycosylation occurs most often in mucins, which are proteins that areabundant at the exterior of mucosa.
Proteolytic processing?
Proteins can undergo proteolytic processing as a post-translational modification to adopt their functional form.
* This involves the cleavage of specific peptide
bonds in the polypeptide chain at specific sites
that are recognized by the enzymes that break
the bonds.
Where is proteolytic processing common?
Proteolytic activation is common in proteases (protein-cutting enzymes), which are often synthesized in inactive forms, with the active
forms released by proteolysis.
- Hormones are often produced as larger proteins that undergo proteolytic processing. Insulin is an example shown here
N-terminal acetylation?
Proteins can be acetylated at the N-
terminal amino group.
* An acetylated N terminal amino acid
is shown.
* This is most common in intracellular
proteins.
* The acetyl group changes the charge
of the protein, since the positive
charge on the N terminus is lost. It
also can change the folding features
of the protein and its stability.
Hydroxylation?
Proline and lysine can be
hydroxylated in proteins.
* The addition of the polar
hydroxyl group makes an
additional hydrogen bond
possible in hydroxylysine and
makes the aliphatic proline into
a polar hydroxyproline residue.
* This is an important post-
translational modification in
collagen, which we will see
again shortly.
Two kinds of post-translational modification can occur on Thr residues (depending on sequence etc.). The are:
A. Phosphorylation and O-linked glycosylation
B. O-linked glycosylation and N-linked glycosylation
C. N-linked glycosylation and hydroxylation
D. Hydroxylation and phosphorylation
E. None of the above.
A) Phosphorylation and O-linked glycoslyation
Typical antibody?
A typical antibody is Immunoglobulin G (IgG).
Features include:
- variable antigen binding sites
- antigen binding regions
- complement regions
- stabilized by intra and inter chain disulfide (s-s) bonds.
- IgG consists of two heavy (50kDa) and two light (25kDa) chains.
- This protein has quaternary structure and is a heterotetramer.
Domains of IgG?
Immunoglobulins and related proteins contain many immunoglobulin domains.
Epitopes?
Antibodies bind to small surfaces if antigens that are called epitopes (very small and specific)
Immunochemical detection?
Antibodies bind an epitope by having a complementary surface (right shape), and stabilizing interactions (non-covalent such as H-bonds, electrostatic forces and the hydrophobic effect) with the epitope.
How to detect or quantify antigens?
Antibodies are used to detect and quantify antigens, either directly (e.g. ELISA) or following SDS-PAGE (western blotting).
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