Aggregated Proteins

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AGGREGATED PROTEINS


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Protein aggregation is a biologicalphenomenon in which mis-folded proteins aggregate (i.e., accumulateand clump together) either intra- orextracellularly
http://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Proteins


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Protein Aggregation

Numerous physicochemical stresses can induce protein aggregation:

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


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Protein Aggregation

Stress (environmental)induced misfolding generates

sticky aggregation proneconformation

Normally folded proteininteracts with misfolded

protein

Cycle multiplies copies ofmisfolded proteins

Normally

foldedprotein

Misfoldedprotein

Large aggregates and fibrils

Oligomers


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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 transmembraneregulator

Sickle cell anemia Hemoglobin

Hungtington disease Huntingtin

Creutzfeldt-Jakob disease Prion protein

Amyloidosis Ten other proteins


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Implication of protein misfolding

1. Gain of toxicityThe harmfull effect of the misfolded protein may be due to deleterious gain offunction as seen in many neurodegenerative disorders (Alzheimer disease,Parkinson disease, Hungtington disease), in which protein misfolding results inthe formation of harmfull amyloid. Neurodegenerative diseases are characterized

by the accumulation of misfolded proteins and formation of aggregates

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

3. AccumulationProtein aggregates are sometimes converted to a fibrillar structure. Fibrilsthemselves are not toxic but insoluble. Their accumulation cause tissue damage(amyloidoses)


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Polyethylene glycol(PEG 3350)

0.1-0.4 g/L L-Arginine hydrochloride 0.4-0.8M

Nondenaturatingconcentrations of Urea

< 2.0 M K-Glutamate ~5M

Nondenaturatingconcentrations Gdm/ClH

< 1.0 M 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

Reagents used for Re-folding of proteins

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


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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

Na2SO

4 or K

2SO

4 0.4-0.6 M

Dodecyl Maltoside 2.0-5.0 mM

Cyclodextrin 20-100 mM Sarkosyl 0.05-0.5 %

Reagents used for Re-folding of proteins (Continued)

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


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Chaperonesassist 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 hydrophobicsurfaces 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 thecell, stabilize proteins against thermal and chemical denaturation(glycerol).

Pharmacological chaperones bind to specific conformations and stabilizethem. They are effective in rescuing proteins from proteasomaldegradation.


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Thermophilic Proteins Living organisms can be found in the most unexpected places,

including deep sea vents at > 100 C and several hundred barspressure, in hot springs, and most recently, deep in the bowels ofthe earth, living off H 2 formed by chemical decomposition of rocks!

The proteins found in thermophilic species are much more stable

than their mesophilic counterparts (although this corresponds toonly 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.


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Protein Stability

Protein stability is the net balance of forces,which determine whether a protein will be inits native folded conformation or a denaturedstate.


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Chemical Stability

Chemical stability involves loss of integrity due to bondcleavage. 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 disulfideinterchange and oxidation of cysteine residues.


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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 andprotein drugs.

Two relatively recent innovations, which have had

major impact in the study of the thermodynamics ofproteins were the development of very sensitivetechniques, differential scanning calorimetry(especially by Privalov and Brandts) and site-directed

mutagenesis.


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Stability of the Folded State

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


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Techniques for Measuring Stability

Any methods that can distinguish between U and FAbsorbance (e.g. Trp, Tyr)Fluorescence (Trp)-difference in emission max &

intensity.CD (far or near UV) - (2 o or 3 o)NMRDSC (calorimetry)Urea gradient gelsCatalytic activityChromophoric or fluorophoric probes

Aggregated Proteins

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