Aggregation Flashcards
Inside of a folded protein is […]
largely hydrophobic
Proteins are only […] stable.
i.e., their native structures
Marginally thermodynamically
Why are proteins only marginally thermodynamically stable?
- Hydrophobic effect (increase in entropy of water upon burial of the hydrophobic groups)
- Penalty of conformational entropy of protein (Decrease in entropy)
- Net stability of the protein is relatively small due to the opposing forces.
What are the three fundamental types of protein stability?
- Thermodynamics (direction of change; population states at equilibrium)
- Kinetics (time scale; rate of change)
- Protease resistance (thermodynamic and kinetic stability are irrelevant if the protein can be broken down by a protease)
What determines kinetic stability of a protein?
Energy barrier
* Folding/unfolding rates
* Kinetic stability/shelf-life
* Resistance to aggregation
What does it mean for a protein to be kinetically trapped?
- A protein is said to be kinetically trapped when it exists in a folded or misfolded conformation that is not at its global free energy minimum, but cannot easily transition to a lower-energy state due to a high energy barrier.
- This concept arises in the context of protein folding and stability, where the “kinetically trapped” state may be stable over long periods due to the significant time or energy required to overcome the barrier separating it from its most stable (native) conformation.
Conformational energy landscapes govern […]
protein structural transitions
What are protein structural transitions?
- Denaturation
- Unfolding
- Aggregation
- Precipitation
- Gelation
- Ligand binding (substrate; co-factor; inhibitor)
- Protein-protein binding
- Protein binding to DNA, lipids, CHO
Protein folding occurs in two steps.
True or False?
True and False - depends on the protein.
Describe protein folding on a funnelling energy landscape.
Describe conformational energy landscapes in food processing.
Describe the cartoon landscape of aggregated proteins compared to proteins in their native state.
The aggregated state is […] than the native state.
More stable
High energy intermediate transition states exist at the top of each barrier.
What are prions?
A special case of protein aggregation
List some protein misfolding diseases. [11]
- Alzheimer’s (Aβ peptide)
- Parkinson’s (α-synuclein)
- Amyotrophic Lateral Sclerosis (SOD1)
- Tauopathies (Tau): common in athletes that experience head trauma (even mild like head-butting the soccer ball)
- Systemic amyloidosis (lysozyme, transthyretin, serum amyloid A)
- Huntington’s (polyQ-rich proteins)
- Type II diabetes (amylin)
- Dialysis-associated (β2-microglobulin)
- Light chain amyloidosis (IgG)
- Transmissible spongiform encephalopathy (PrP)
Describe the age-specific prevalence of Alzheimer’s Disease.
What is transmissile spongiform encephalopathy?
- Affects animals (e.g., cattle; deer; moose; elk; sheep; people)
- Can be acquired by humans by eating infected flesh
- Food safety risk
- 100% fatal
- No cure
- Pathological agent = protein aggregates
Prion disease
Compare the native and abnormal prion protein.
- Native prion protein (PrP): found in all body tissues - predominantly in the brain
- Abnormal conformation (“prion”): disease occurs when normal PrP is converted into a particular conformation
What is a prion?
- Withstands heat, UV radiation, nucleases
- Free of nucleic acid (RNA; DNA)
- Not due to virus, bacteria, fungi, or parasites
Infectious protein
Conversion mechanism is unknown; structure of infectious/toxic prion is unknown
Describe the conversion of native PrP into prion.
- What is the structure of this prion? (monomer? dimer? amyloid?)
- How is prion formed?
What are the features of prion disease? [11]
Transmissible Spongiform Encephalopathy
- Transmissible
- Oral route of infection
- Survive metabolic clearance
- Self-replicates in the body
- Reach target organ (brain)
- Induce cascade of neurodegeneration
- Long incubation period (2-10 years)
- Death occurs within weeks-months of symptoms
- 100% fatal
- No treatment
- Different strains exist that lead to clinically distinct diseases
What are the names and routes of infection for the 6 human prion diseases?
- Kuru: canabalism
- Creutzfeldt-Jakob disease: sporadic; genetic; acquired
- Variant CJD: ingested BSE
- Gerstmann-Straussler-Scheinker syndrome: genetic
- Fatal familial insomnia: genetic
- Sporadic fatal insomnia: genetic
Describe cases of BSE (mad cow disease) in the UK.
- 180,000 positive cases
- 4.4 million animals culled
- 0.5 million infected animals entered human food chain
Describe how BSE was linked to vCJD.
- Silent carriers: 1/2000 Britons carry abnormal prion in appendix, tonsils
- Uncertain as to whether a second cohort of vCJD victims will appear
- People from UK and France not allowed to donate blood in Canada
How was the BSE epidemic created?
Unfortunate feeding practices
How was the BSE epidemic in the UK controlled?
Meat bone meal banned from animal feed
Describe BSE in Canada and the USA.
First domestic case in 2003 - a single cow shut down beef exports for years. Huge losses. “Shoot/shovel/shut-up!” - says Alberta premier at the time. (not a great idea since the proteins can survive easily in soil)
What are specified risk materials in Canada, regarding BSE?
- The skull, brain, nerves attached to the brain, eyes, tonsils, spinal cord & nerves attached to the spinal cord of cattle aged 30 months or older.
- The distal ileum (portion of small intestine) of cattle of all ages.
Describe the distribution of Chronic Wasting Disease in North America.
Describe cases of Chronic Wasting Disease in BC.
Are there any anti-aggregation compounds, nutraceuticals, or functional foods?
- Small molecules that are known to bind (at least in vitro) to proteins and prevent them from aggregating or change how they aggregate.
- They are not specific to prions - they will bind to proteins rich in aromatic amino acids through their aromatic functional groups - via pi-stacking.
What is the possible role of anti-aggregation compounds to prevent prion formation?
- General mechanism for binding of polyaromatic compounds to proteins (e.g., tea polyphenols bind beta-lactoglobulin milk protein) is π–π stacking interactions
How does PrP convert into prion?
Why are details of prion formation obscured?
Ensemble averaging
You do not see what each individual molecule is doing, just the average of all of them.
What is the single molecule approach?
Detect ‘invisible’ states; map out pathway
This may be useful because details of prion assembly are obscured by ensemble averaging.
How can singular molecules be studied?
Using Optical Tweezers
Hamster prion disease shown in these graphs.
Plastic beads and DNA are used to pull a protein apart.
Compare native and misfolding energy landscapes.
PrP + PrP = always misfold!
What is the role of glycosylation in conformational strains of prion?
Strains = different conformations of the disease form, PrP^Sc
Inducing PrP fibrils in vitro:
* Chemical denaturant
* Shake/rotate/stir
* Reducing agents
* Low pH
Describe the role of glycosylation in prion protein structure.
- Believed to play key role in strain & species barriers
- Structural/biophysical studies used non-glycosylated PrP
- Prions may be unglycosylated, or glycosylated with 1 or 2 glycans.
* Play a role in receptor-mediated entry into tissues (e.g., perhaps this is why some cross the blood-brain-barrier)
