Protein Folding &

Chaperones
 Factors influencing protein activity

 Denaturation Vs. inactivation

 Denaturation ……Random coil: no specific shape:
Physical, Biological or Chemical
 Is it reversible (some cases)?
 Inactivation/activation: Form of regulation may be
reversible or irreversible.
Native Form and Denaturation
 Inactivation

 Covalent modification
Reversible: phosphorylation/dephosphorylation
 Acylation ex: acyl group (C16-C14) : Myristoylation,
palmitoylation
 Prenylation (isoprene 5 C) (hydrophobic)
 Allosteric modulators positive and negative, Ca (+ATP)
 Conformational: (dimer of dimers, bind O2 # affinity )
monomer - polymer
 Covalent: Irreversible: Proteolysis
 Denaturing Agents

 Harsh mechanical treatment/handling (Ptn

albumin)
 Heat : (Ionic interaction & H Bonding)
How does changes in temperature affect protein
function?
 Heat Kinetic energy
 (Taq-polymerase), freezing & thawing
Acid-base: Charge disruption-Amide-H
bonding
Alcohol (H bonding)
 Denaturating agents

 Salt concentration, Heavy Metal (Strong Electrophiles), Cu,

Pb & Mercury
+NH > k> + Na + > Li + > Mg++ > Ca++ precipitating
4

Urea solubilizing Guanidium

 Disulfide reduction
B-Mercaptoethanol, Dithiothreitol (Clelalnd)

β-mercaptoethanol, BME, 2BME, 2-ME or β-met

Dithiothreitol (DTT))
Detergents (Surfactant): Ionic & NonIonic

• amphipathic molecules, containing a polar hydrophilic head group

attached to a long-chain hydrophobic carbon tail.
• non-denaturing detergents (Non Ionic), separate proteins

NonIonic
oxyethylene polymers (e.g. Brij® and TWEEN®) or ethyleneglycoether
polymers (e.g. TRITON®)
TRITON X-100 and IGEPAL® CA-630, have an aromatic head
 Ionic Detergent (whole cell Lysis)

SDS: (Harsh detergent)

Sodium Dodecyl Sulfate, Sodium Lauryl Sulfate

At a sufficiently high concentration, the polar

hydrophilic region of each molecule is oriented toward
the polar solute (water) while the hydrophobic regions
are grouped together to form thermodynamically
stable micelles with hydrophobic cores
 Other denaturing agents

 Heavy metals: Pb, Hg, Cd, Ag

 UV ionizing radiation

 Will a denatured protein re-nature into its native

conformation?
Unfolding: Folding

Performic acid----Sulfonic Acid:

Key Point in folding: Hierarchy:

 Protein folding

 IS IT RANDOM? In complex system duplicated

time after time
 What forces direct the folding? (Environment and
Protein itself)
 Gibbs Free energy
 Protein folding

 Gibbs Free energy or available energy

 Thermodynamic potential measuring usefulness of a
process extracted from a closed system.
 ∆G = ∆H - T∆ S
 T ∆ S : entropy (Disruption, highly positive since the
amide bond are forced to fold and are forced together
aside); T: Kelvin: is the measure of disorder: the
tendency of a system to become random. It increases in
spontaneous reactions. : less random entity is actually
invested by its surrounding.
 ∆ H: Enthalpy : total energy of thermodynamic system
 ∆ G : is the work done exchanged by the system and its
surroundings
 Folding Pathways

 Not random.
 The folding process is rapid, dictated,
 Determined by the primary sequence and
surrounding environment,
 Requires sometimes accessory proteins (refold in
vitro, requires long time, even end up aggregating ).
 Protein Folding

 Ribonuclease A (RNase A) will refold to

native structure spontaneously (1 minute)
 >1050 possible conformations

 If 10-13 sec per conformation, it would

take 1030 years to sample enough to
determine structure
 How do proteins fold so quickly?
 Accessory Proteins

 Chaperons
 Prolyl Cis –trans isomerase
 Protein Disulfide Isomerase…… PDI
 Calnexins
Accessory Proteins (Chaperons)

 Chaperons, Chaperonines & HSP:

 Ptns that bind to newly synthesized peptide sequences
and assist in 3D formation of new peptide;
 prevent unwanted binding
 Ubiquitous,
 Assist in assembly & refolding
 Requires Energy (ATP)
 In cases of Stress upregulated and are upregulated
 HSP

 HSP 70 ……. Blocks aggregation, Phobic binding ,

protect from solvent –assisted with HSP-40

 HSP60 …… Provides isolation chamber, prevent

aggregation, phobic inside
 Calnexins

 Calcium binding proteins located in the ER

membrane,
 Binds monoglycosylated species of the glycoproteins
 Retain them in the ER until glycoprotein has folded
properly
Prolyl cis-trans isomerase
 Disulfides Bonds

 Stabilize native structure

 Formed after native 2ndry conformation achieved
 Abundant in secreted proteins but not in intracellular
proteins
 Protein disulfide isomerase catalyzes reduction of
incorrect disulfide linkages
 Protein Disulfide Isomerase (PDI)

 Catalyzes disulfide exchange: rupturing improper S-S

Bonds and reformation with exchange with a partner
 Protein Fate:
Any Clue where are we Going 
 Protein Misfolding

Protein Conformational Diseases

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

Loss of function or gain of toxic activity

PFD
 Homework

 Comparison among different detergents and Mode of

Action in Protein Lysis (Tris-HCL & HEPES)?
 DTT
 MercaptoEthanol
 SDS
 RIPA Lysis buffer
 Discuss Gibbs free energy ?
 Enthalpy: Entropy & Relation to Protein Folding and
 how Protein Folding spontaneous
 How protein folds thermodynamically?
 Non Ionic & Ionic Detergent