Protein Folding, Misfolding, Aggregation, and Disease Flashcards
Can form aggregates that interfere with other cellular functions
Unfolded Proteins
Regulatory mechanisms that promote correct folding are balanced by proteolytic pathways that degrade persistantly damaged
Proteins
Increased levels of misfolded proteins can lead to a number of disease, including
Neurodegenerative conditions
Can play a significant role in guiding the correct folding of the polypeptide chain, to generate the structural and catalytic properties of the protein
The sequence of amino acid residues
While genetic mutations affect all the polypeptide chains produced from a specific mRNA, errors can also arise from inserting incorrect amino acid in the growing chain, and by slippage of the ribosome on the mRNA template resulting in
Frameshifting
Can occur spontaneously, or be induced by environmental stresses
Post-translational misfolding
Can occur co-translationally or post-translationally and can confer alternate biochemical fates
Protein folding
What interactions promote protein folding?
- ) Hydrophobic
- ) Electrostatic
- ) van der Waals
- ) Disulfide bonds
- ) Metal coordination
What are some agents that promote unfolding?
Temperature, pH, pressure, urea, etc
Key information for protein folding is present in the
Polypeptide sequence
Based on a fundamental understanding of the physical and chemical properties of amino acids, proposed the formation of α-helices and β-sheets
Linus Pauling
Showed that bovine pancreatic ribonuclease could be fully denatured by treatment with β-mercaptoethanol (β-Me reduces disulfide bonds) and 8M urea (which unfolds proteins by disrupting non-covalent interactions and solubilizing non-polar residues).
Anfinsen experiment
In the Anfinsen experiment, rapid removal of β-Me and urea only allowed
1% of activity of protein to be recovered
In the Anfinsen experiment, slow removal of β-Me and urea by step-wise dialysis restored
Almost full activity
The conclusion from the Anfinsen experiment was that the information for generating the secondary and tertiary structure in a protein is intrinsically available in the
Polypeptide sequence
All the information necessary to assemble and generate full enzymatic activity is present in the
Amino acid sequence
Small proteins may fold rapidly and spontaneously, however, large proteins tend to require
Chaperones
States that particular folding pathways must be favored by a specific protein because otherwise it would take too long for proteins to fold
Levinthal’s Paradox
Do not enhance correct folding, rather they prevent non-productive routes
Molecular chaperones
Posits that evolution has selected polypeptide chains in which the individual amino acids are positioned so that they maximize correct folding events, and minimize structural barriers (through their side chains).
Bryngelson and Wolynes Principal of Minimal Frustration
Bryngelson and Wolynes Principal of Minimal Frustration basically says that the folding pathway for a polypeptide does not proceed in a
Linear manner
The ΔG between unfolded and folded/native protein is very
Small
The average stability per residue is
0.1 kcal/mole
Can promote folding of specific proteins, or participate in general quality control mechanisms
Chaperone proteins
Chaperone proteins that promote the folding of proteins emerging from the ribosome are likely to be
Non-specific
Chaperone proteins that assist in the assembly of large multi-subunit complexes are typically
Highly specific to a particular task
Has a significant impact on the global structure of the folded protein
Hydrophobic core
Have a significant impact on the local environment of a folded protein
Electrostatic interactions and van der Waals interactions
Have a significant impact on a folded proteins stability
Disulfide bonds
Has a significant impact on the structure and stability of a folded protein
Metal coordination
A common DNA binding domain present in many enzymes and nucleic acid binding proteins
Zinc Finger (RING motif)
Water molecules around a hydrophobic protein structure are constrained because certain hydrogen bonds resist pointing towards the
Hydrophobic amino acid residues
Larger proteins that achieve 3-D conformation with help from a chaperonetypically can not be
Renatured following denaturation
Protein denaturation is not concentration dependent (zero order kinetics) and is simply a function of protein
Vulnerability to the denaturant
The temperature at which 50% of the proteins molecules are unfolded
-A measure of the thermodynamic stability of the protein
Transitional melting Temperature (Tm)
The Tm can be affected by
Mutation, post-translational modification, and association with other proteins
What are four agents that can promote folding?
Co-factors, disulfide bond, chaperones, and physiological partners
Help to shield the hydrophobic cores of proteins until they are released from the ribosome and can fold into their final conformations
Molecular chaperones
The chaperones stabilize the nascent chain, prevent deleterious interactions with other constituents in the cell, and provide an opportunity for the protein to achieve its
Mature structure
Predicts that folding resembles a simple chain reaction with reactants and products
-Each sub-step culminates in a folded intermediate that can then proceed into the next folding event
Sequential folding model
The sequential folding model is improbable since
Intermediates have never been detected
Posits that a polypeptide chain can enter multiple folding pathways, although only one path leads to a productive native structure
Continuum folding model
A polypeptide chain that has achieved a near-final secondary structure (i.e. alpha helices & beta sheets), but is maintained in a less ordered 3-D structure that is ‘looser’ and more ‘open’ than the final structure
Molten Globule
Not a defined structure, but refers to a family of related structures that are fluid and interchangeable
-Can be represented as U M N
Molten Globule
The driving force in the molten globule is
Water exclusion
Related to the observation that ‘off-pathway’ folding events are not energetically prohibited. In fact, the energy difference between an on-pathway structural intermediate and an off-pathway structure is only -10 kcal/mol
The need for Chaperones
Increase the likelihood of guiding ‘on-pathway’ folding intermediates, ultimately leading to mature and
functional proteins
Molecular chaperones
Very similar in structure, each containing a barrel with four rings
Proteosome and GroEL chaperone
Chaperones use ATP hydrolysis to generate energy and turn it into torque to refold the protein. This requires the hydrolysis of
14 ATP
Protect the nascent chain and give guidance during folding to prevent kinetic dead ends
Chaperones
Chaperones sitting by the ribosome decide if a protein is folding properly or if it needs to be
Degraded
Structurally similar to the chaperone, but its hydrophobic channel is much more narrow, which prevents folded proteins from entering
Proteosome
The proteosome is made up of four rings, 2 α and 2 β, and three of these subunits are made up of
Zymogens
Consumed in proteosome formation
UMP 1
What are the three modules in protein folding?
Hierarchical, Nucleation-condensation, and Hydrophobic collapse
Secondary structures form first, and then through intramolecular interactions promote the assembly of the 3-dimensional structure. In the absence of the 3-dimensional interactions, the secondary structure is not stable
Hierarchical module
Local sites of structural nucleation results in rapid propagation of the structure motif, which coalesce and stabilize the final native structure
Nucleation-Condensation module
The molten globule forms as a result of tertiary hydrophobic interactions that initiate secondary structure maturation and the 3-D structure
Hydrophobic collapse module
Cleaves insulin into it’s final conformation
Carboxypeptidase e
Initially interact with short hydrophobic patches in nascent polypeptide chains, and form a stronger binding with the hydrolysis of ATP.
Cytosolic Hsp70 proteins
These cycles of binding and release are coupled to improve folding of the emerging chain, in part by preventing
Non-productive endpoints
Do NOT increase the rate of folding reactions, but DO improve the yield of successfully folded products
Chaperones
Overcome energetic barriers that slow specific folding steps and can thereby increase folding rates
Protein disulfide isomerase and prolyl isomerase
Can transiently bind unfolded segments during the translocation of proteins to the ER and mitochondria, which requires partial unfolding.
Cytosolic Chaperones (Hsp70’s)
Also present in the lumen of the ER to facilitate refolding and complex assembly
Chaperones
Fix improperly formed disulfide bonds and ensure that the correct cysteine residues are paired together
Disulfide isomerases
This mutation in the cystic fibrosis transmembrane receptor proteins causes slow folding into the 3D structure. The protein is thus captured by the proteolytic system and degraded.
CFTRΔ508
Plays a critical role in recruiting charged
tRNA’s to the ribosome, and in ensuring that the correct anticodon::codon pairing ensues. However, when a polypeptide chain fails to fold correctly, it facilitates the degradation of the unfolded chain.
eEF1A
In order to form, the proteosome requires
4-5 chaperones
Plays an important role in targeting unfolded ER proteins to the cytosolic protein degredation pathway
Endoplasmic Reticulum Associated protein Degredation (ERAD)
Promotes the assembly of components of the mitochondrial energy generating pathway
-Can be inhibited by geldenamycin
Hsp90
The ribosome error rate is
1 out of every 10^4 aminos incorporated
A multi-subunit cylindrical particle consisting of four rings that can bind unfolded proteins in large hydrophobic cavities present at both ends of the cylinder
-Hydrolyzes 14 ATP per protein
GroEL chaperone
The GroEL chaperonin consists of seven identical subunits, which have three distinct domains, termed equatorial, intermediate, and axial. The axial domain forms a large cavity lined with hydrophobic residues that bind
Unfolded Proteins
Once the unfolded protein has been captured, the GroEL cavity is covered by the
-Prevents premature release of the substrate
GroES complex
When GroEL hydrolyzes ATP, it turns, exposing hydrophilic residues in the cavity which cause
Hydrophobic aminos of the protein to be buried in the center (promoting folding)
Most neurodegenerative conditions (including Parkinson’s, Alzheimer’s, and Amyotrophic Lateral Sclerosis) are associated with high levels of
Insoluble proteins (Aggregates)
Protein folding also plays a key role in diseases such as mad cow disease and Creutsfeldt-Jakob Disease (CJD), which are both
Prion Diseases
Prion diseases can be caused by a mutation in the
PRNP gene
A form of transmissible prion disease, which is acquired through ritual cannibalistic activities
Kuru
Proteosome substrates can come from co-translational protein misfolding, short-lived regulatory proteins, and
Stress-induced protein damage
A large clump of damaged, unfolded proteins all in one area
Aggresome
The amyloid diseases share a common feature of accumulating fibrous plaques containing mostly
β-pleated sheet aggregates
Protein infectious units that can cause chain reactions of unfolding amongst wild type proteins
Prions
Characterized by the accumulation of aberrant Aβ peptide in the plaques
Alzheimer’s Disease
CAG repeats (glutamine) that undergo extensive expansion to cause altered function or aggregation. Ex: Huntington’s Disease, Machado Joseph Disease
Trinucleotide expansion diseases
