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AGGREGATED PROTEINS

Protein aggregation is a biological


phenomenon in which misfolded proteins aggregate (i.e., accumulate
and clump together) either intra- or
extracellularly

Protein Aggregation

Numerous physicochemical stresses can induce protein aggregation:


Heat, pressure, pH, agitation, freeze-thawing, dehydration, heavy metals,
phenolic compounds, and denaturants.

Protein Aggregation
Stress (environmental)
induced misfolding generates
sticky aggregation prone
conformation

Normally
folded
protein

Normally folded protein


interacts with misfolded
protein
Cycle multiplies copies of
misfolded proteins
Misfolded
protein

Oligomers

Large aggregates and fibrils

Diseases

Protein involved

Alzheimers disease

Amyloid-

Parkinson disease

-Synuclein

Diabetes type 2

Amylin

Amyotrophic lateral sclerosis

Superoxide dismutase

Haemodialysis-related amyloidosis 2-microglobulin


Cystic fibrosis

Cystic fibrosis transmembrane


regulator

Sickle cell anemia

Hemoglobin

Hungtington disease

Huntingtin

Creutzfeldt-Jakob disease

Prion protein

Amyloidosis

Ten other proteins

Implication of protein misfolding


1. Gain of toxicity
The harmfull effect of the misfolded protein may be due to deleterious gain of
function as seen in many neurodegenerative disorders (Alzheimer disease,
Parkinson disease, Hungtington disease), in which protein misfolding results in
the formation of harmfull amyloid. Neurodegenerative diseases are characterized
by the accumulation of misfolded proteins and formation of aggregates

2. Loss of function
Other effect of the misfolded protein may be due to loss of function, as observed
in cystic fibrosis. There is a mutation in the CFTR sequence

3. Accumulation
Protein aggregates are sometimes converted to a fibrillar structure. Fibrils
themselves are not toxic but insoluble. Their accumulation cause tissue damage
(amyloidoses)

Reagents used for Re-folding of proteins

Polyethylene glycol
(PEG 3350)

0.1-0.4 g/L

L-Arginine hydrochloride

0.4-0.8M

Nondenaturating
concentrations of Urea

< 2.0 M

K-Glutamate

~5M

Nondenaturating
< 1.0 M
concentrations Gdm/ClH

Proline

~1M

Methylurea

1.5-2.5 M

Glycerol

20-40 %

Ethylurea

1.0-2.0 M

Sorbitol

20-30 %

Formamide

2.5-4.0 M

Sucrose

~1M

Methylfomamide

2.0-4.0 M

Trehalose

~1M

Acetamide

1.5-2.5 M

TMAO
(trimethylamine N-oxide)

~1M

Ethanol

Up to 25%

Sulfo Betaine

~1M

(http://www.ls.huji.ac.il/~purification)

Reagents used for Re-folding of proteins (Continued)

n-Penthanol

1.0-10.0 mM

Lauryl Maltoside

0.06 mg/ml

n-Hexanol

0.1-10.0 mM

CETAB

0.6 mg/ml

Cyclohexanol

0.01-10.0 mM CHAPS

10-60 mM

Tris

> 0.4 M

Triton X-100

10 mM

Na2SO4 or K2SO4 0.4-0.6 M

Dodecyl Maltoside

2.0-5.0 mM

Cyclodextrin

Sarkosyl

0.05-0.5 %

20-100 mM

(http://www.ls.huji.ac.il/~purification)

Chaperones

assist other proteins to achieve a functionally active 3D structure


prevent the formation of a misfolded or aggregated structure

Molecular chaperones recognise misfolded protein, bind to the hydrophobic


surfaces and inhibit aggregation. Most of these molecules are heat shock
proteins (formed during thermal damage)-protect against denaturation.
Chemical chaperones influence the protein folding environment inside the
cell, stabilize proteins against thermal and chemical denaturation
(glycerol).
Pharmacological chaperones bind to specific conformations and stabilize
them. They are effective in rescuing proteins from proteasomal
degradation.

Thermophilic Proteins
Living organisms can be found in the most unexpected places,
including deep sea vents at > 100 C and several hundred bars
pressure, in hot springs, and most recently, deep in the bowels of
the earth, living off H2 formed by chemical decomposition of rocks!
The proteins found in thermophilic species are much more stable
than their mesophilic counterparts (although this corresponds to
only 3 - 8 kcal/mol of free energy).
The upper limit of temperature growth for bacteria is about 110
C.
Many of the species found in these extreme environments
(T > 100C, pH 2) belong to the Archeae kingdom.

Protein Stability
Protein stability is the net balance of forces,
which determine whether a protein will be in
its native folded conformation or a denatured
state.

Chemical Stability
Chemical stability involves loss of integrity due to bond
cleavage.

deamination of asparagine and/or glutamine residues,


hydrolysis of the peptide bond of Asp residues at low pH,
oxidation of Met at high temperature,
elimination of disulfide bonds
disulfide interchange at neutral pH

Other processes include thiol-catalyzed disulfide


interchange and oxidation of cysteine residues.

Protein Stability
Protein stability is important for many reasons:
Providing an understanding of the basic thermodynamics of
the process of folding,
increased protein stability may be a multi-billion dollar value
the in food and drug processing, and in biotechnology and
protein drugs.

Two relatively recent innovations, which have had


major impact in the study of the thermodynamics of
proteins were the development of very sensitive
techniques, differential scanning calorimetry
(especially by Privalov and Brandts) and site-directed
mutagenesis.

Stability of the Folded State


Measuring protein stability is measuring the energy
difference between the U (unfolded) and F (folded)
states.

Techniques for Measuring Stability


Any methods that can distinguish between U and F
Absorbance (e.g. Trp, Tyr)
Fluorescence (Trp)-difference in emission max &
intensity.
CD (far or near UV) - (2o or 3o)
NMR
DSC (calorimetry)
Urea gradient gels
Catalytic activity
Chromophoric or fluorophoric probes