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What are two examples of post-translational modifications?
Covalent modifications of amino acid side chains
Proteolytic cleavage of inhibitory portion of protein
What are some examples of covalent modifications?
Methylation of Lysines
Acetylation of Lysines
Phosphorylation of Serines
Ubiquitylation of Lysines
What’s the purpose of proteolytic cleavage?
Acts upon mature proteins that are naturally inactive
To activate a protein
To create a signaling cascade (ex. proteases)
How many protein modification sites can a protein have?
Multiple! But not all will be used at the same time!
What is phosphorylation?
Addition of NEGATIVELY CHARGED phosphate group to the R-group of a…
serine
threonine
tyrosine
Where does the phosphate come from for phosphorylation?
From ATP!
The phosphorylated amino acid + ADP is formed
What catalyzes phosphorylation?
Protein kinases!
In charge of adding a phosphate!
Is phosphorylation reversible?
Yes, BUT not hydrolysis!
We can remove the phosphate from the protein but do not recover energy.
What removes the phosphate from proteins after phosphorylation?
Protein phosphatases!
What’s the effect of phosphorylation?
Makes proteins with a NEUTRAL charge into NEGATIVE charge!
Major structure changes, activity changes, or changes in protein solubility
Serve as a recognition site (other proteins can bind to it)
Each phosphate group adds two negative charges to the protein!
What is ubiquitylation of lysines?
The addition of ubiquitin!
Covalently attach a large tag of amino acids! Only modification that is an actual protein.
What’s the function of ubiquitylation?
Marks proteins for degradation
Direct proteins to specific locations in the cell (localization)
What are ligands?
The molecules that proteins bind to!
Is ligand binding reversible?
Yes! More than covalent modifications!
What factors must be considered in the strength of protein binding?
Interactions must be strong
to resist the bouncing from random movements
to resist environment molecules that bump into it
How is binding strength achieved?
3D complementary binding (grooves match perfectly)!
formation of several noncovalent bonds (strength in numbers)
Why is correct folding for protein function essential?
Amino acids that contribute to binding a ligand are often quite far apart on a protein’s primary sequence!
What is Kon?
The rate of the forward (association) reaction!
What is Koff?
The rate of the backward (dissociation) reaction!
What is Ka?
The association constant! Kon / Koff
Measures the strength of binding
What is Kd?
The dissociation constant! 1/Ka
MUCH EASER TO CALCULATE and can also measure strength of binding
Meaning of large and small Kd values?
Larger = small Ka value = weaker binding
Smaller = larger Ka value = stronger binding
What can Kd also measure other than binding strength?
Can measure concentration of ligand required to ½ saturate protein!
What is regulation by direct phosphorylation?
SOURCE OF PHOSPHATE is ATP
Protein kinase takes P from ATP and phosphorylates the protein!
Protein is then dephosphorylated by protein phosphatase.
REPEAT.
Exception of ATP hydrolysis for REGULATION.
What is regulation by binding to phosphorylated/dephosphorylated forms of another nucleotide?
GTP binds to the protein which activates it.
The phosphates cleaves off and leaves GDP attached to the protein.
GDP then also leaves binding site turning it off.
REPEAT
What are GEFs?
Guanine Nucleotides Exchange Factors!
Pry open protein so GDP falls off and allows GTP to enter the binding site (GTP is found in higher amounts).
What are GAPs?
GTPase activating proteins
Negative regulators that cause the hydrolysis of GTP (phosphate leaves)!
What are Cyclin Depedent Kinases (CDKs)?
Exhibit cell cycle regulation!
Activity regulated by…
Phosphorylation at one site (to activate)
Dephosphorylation at another site (to deactivate inhibitor)
Binding of the protein cyclin
Note 
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