What factors influence protein function?

What is denaturation ?
What is inactivation?
What forces underlie the folding process?
What are the consequences of protein misfolding?
Factors influencing protein activity
Denaturation Vs inactivation
Denaturation Random coil: no specific shape.
Is it reversible?
Inactivation/activation form of regulation may be
reversible or irreversible.

In/activation
Covalent modification
Reversible: phosphorylation/dephosphorylation

Irreversible: Proteolysis
Acylation ex: Myristoylation, palmitoylation
Allosteric modulators positive and negative
Conformational: monomer - polymer
Denaturing Agents
Harsh mechanical treatment/handling
Heat
How does changes in temperature affect protein
function?
Heat Kinetic energy
(Taq-polymerase), freezing and thawing


Acid-base: Charge disruption
Alcohol
Denaturating agents
Salt concentration
+
NH
4
> k>
+
Na
+
> Li
+
> Mg
++
> Ca
++
precipitating

Urea solubilizing Guanidium











Disulfide reduction
Mercaptoethanol, Dithiothreitol

Detergents:

SDS:
Sodium dodecyl sulfate, Sodium lauryl sulfate
Other denaturing agents
Heavy metals: Pb, Hg, Cd, Ag
UV ionizing radiation


Will a denatured protein re-nature into its native
conformation?
Anfinsen Experiment
Protein folding
IS IT RANDOM?

What forces direct the folding?

G = H - T S











Folding Pathways

Not random.
The folding process is rapid
Determined by the primary sequence and surrounding
environment,
Requires sometimes accessory proteins.








Accessory Proteins

Prolyl cis trans isomerase


Protein Disulfide Isomerase PDI


Chaperons


Prolyl cis-trans isomerase
Protein disulfide Isomerase (PDI)
Chaperons
Heat shock proteins

HSP 70 . Blocks aggregation

HSP60 Provides isolation chamber
Protein Fate
Protein Misfolding
Protein Conformational Diseases


in 2
ry
and/or 3
ry
structure of functional protein


Loss of function or gain of toxic activity
PFD
PrP
C
/PrP
Sc

1. PrP
Sc
bind to PrP
C

2. Leads to Flipping
of PrP
C
into a beta-
sheet conformation
(PrP
Sc
)
3. PrP
Sc
can Polymerize
4. Lead to the formation
of fibrils and plaques
5. Deposit in brain
Sickle hemoglobin