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 +NH4 > 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 2ry and/or 3ry structure of functional protein

Loss of function or gain of toxic activity

PFD

PrPC/PrPSc

1. PrPSc bind to PrPC

2. Leads to “Flipping”

of PrPC into a beta-

sheet conformation

(PrPSc)

3. PrPSc can Polymerize

4. Lead to the formation

of fibrils and plaques

5. Deposit in brain

Sickle hemoglobin