Topic 4

4A
Post-translational Modifications and Ligand binding

Topic 4: Protein Function

■ Protein function underlies almost all cellular events
■ There are several hundred thousand unique proteins in our bodies!
■ Post-translational modification (PTM’s)
● Huge impact on protein function
● Main contributor to the diversity of protein function

↳*** don't need to memorize this chart

Post-translational modifications (PTM’s)

↳*** Don’t need to memorize all of the PTM’s or the diagram
⇒ Diagram illustrates how various PTM events can regulate the function of a particular protein
(the tumor suppressor suppressor protein (or p53 gene))

■ Phosphorylation
■ Acetylation
■ Hydroxylation​ (not shown in diagram)
↳ Main 3 kinds of PTMS
↳ involve covalent addition of various kinds of molecules to amino acid side
chains
↳ Involve functional group modifications

■ Ubiquitination
■ Sumoylation
↳ Involve the addition of whole proteins to side chains

■ Glycosylation
↳ Involves the addition of sugars to side chains

■ Myristoylation
 ■ Farnesylation
↳ Involves​ ​the addition of lipids to side chains

■ Lipoproteins
○ Binds lipids
○ Complexes of proteins and lipids
- Although the lipids are not covalently attached
○ E.g the bad cholesterol LDL
■ Metalloproteins
○ Bind metal ions
○ E.g Taffy enzyme binds zinc ions

■ Hemoproteins
○ Proteins that have an attached heme
○ E.g, Myoglobin and hemoglobin

⇨ Diagram:
● P53 can become acetylated and phosphorylation in response to some sort of cellular
stress
○ This allows p53 to translocate into the nucleus and form a tetramer
- There, p53 works to increase the expression of target genes to respond to
the stress
● P53 is also negatively regulated by a different PTM
○ This PTM is the addition of a series of ubiquitin proteins
- These proteins target P53 for degradation

Key PTM: phosphorylation

■ Side chains​ with an ​OH group
● E.g. side chains in amino acids serine, threonine and tyrosine
● Can be​ phosphorylated ​by enzymes called​ kinases

■ Phosphorylation creates side chains with unique properties

● Side contains two negative charges
○ When the​ phosphoryl groups​ encounters a lysine or an arginine for
example, they can then ​form a salt bridge​ that can ​change​ the ​confirmation
and/or the​ ​activity​ of a protein

■ Phosphorylation acts like an on and off switch

● To turn off the switch, a diff set of enzymes called ​phosphatases​ ​removes the
phosphoryl group to yield the original hydroxyl

Ligand-binding proteins
 ■ Many proteins contain sites to which ligands specifically bind and form a “complex” with
the protein
● Ligand: ​molecule that can form this complex

■ Ligand-binding
● Reversible interactions
○ Thus these are mediated by ​non-covalent bonds

■ Binding occurs by ​multiple weak​ ​or​ a ​few strong​ forces (forces = non-covalent bonds)
● These interactions lead to ​extreme specificity
○ Kind of like a lock and key
○ Every ligand-binding protein requires a specific ligand
- (only one of the thousands of molecules within a cell can usefully
bind to a particular protein

The specificity of ligand binding has been exploited to large effect by pharmaceutical
companies:
➢ Developed​ compounds​ that through ​molecular mimicry,​ can ​bind in​ the
ligand-binding site of​ key target​ proteins.

↳ E.g.​ ​Naproxen:
■ Naproxen is bound into the enzyme COX-2
● This prevents COX-2 from making
compounds that cause pain and
inflammation

Ligand-binding - The Dissociation Constant

■ Ligand binding is just an equilibrium phenomenon like we have previously seen

■ Kd​ (dissociation
​ constant)
➢ Measure of the ​strength​ or ​affinity​ of an interaction
- I.e., measure of the​ affinity of a protein for its ligand
➢ An​ equilibrium constant

➢ Can tell us if a molecule is in high enough concentration to bind to the protein

○ This is of course impacted by the affinity
➢ We can also discern which one of a set of competing ligands will predominantly
bind
○ This is useful if you’re trying to decide what a good dose might be for a
new drug, for example.

Dissociation constants
■ Inverse relationship ​b/w ​Kd​​ and the ​affinity
● High affinity = tight binding = small ​K​d
■ Units of ​K​d​: moles/L (M)

■ Real examples of ​Kd​ ​values for some ligand/protein pairs:

↳*** Don’t need to memorize numbers

➢ The tightest binding is b/w the vitamin biotin and the protein
streptavidin
 ↳*** Must know the info inside red box
➢ These quantities are essential to experimental biochemistry