Alfonso De Simone Flashcards
General Facts about protein folding - introductory level
What did the Anfinsen experiment show us?
What is the Anfinsen experiment? - Showed that the thermodynamic minimum protein conformation is the native state
When left on its own - the protein will fold in a way to reach this thermodynamic minimum
Outline the steps of the Anfinsen experiment
Procedure
- Denature ribonuclease A (4 disulfide bonds) with 8 M Urea and b-mercaptoethanol to totally unfold the protein in a random coil state having no activity
- Removal of Beta-merceptoenthanol and then urea (attempt to renature protein) –> resulted in protein with no function
Why?
If you were to allow the protein to renature in absence of denaturants we would only get 1% enzymatic activity –> Due to the fact that the probability of one of the Cys to form the correct bond upon renaturation would be 1/7 and then that one of the remaining 6 forming the correct bond would be 1/6, etc. Hence, there is 1/105 chance that all the Cys form the correct bond
- But! Further addition of trace amounts of B-mercaptoethanol converts the scrambled form into native pattern explanation –> This allows non-physiological disulphide bridges to be broken allowing the correct bonds to be made and thus the correct protein structure
Conclusion - This shows us that that the native form correspond to the thermodynamically most stable conformation –> lowest in energy.
In the Anfinsen experiment what other method could be used to reduce the time of renaturation of the scrambled protein?
The time of renaturation of the scrambled protein can be reduced by using protein disulphide isomerase (PDI) which catalyses the disulphide bond interchange.
When is a protein most conformationally stable?
Difference between chemical and conformational stability?
What DG is required for a protein to fold into it’s native conformation? What are some examples of favourable and unfavourable process that occur during protein folding?
What are the external factors, outside the realm of the protein, that influence protein folding?
What are the determinants of protein folding?
Determinants of protein folding –> non-covalent interactions, covalent interactions, compaction, hierarchy, adaptability and sequence versatility.
Why has evolution selected for an average stability around 5 to 10 kcal/mol?
The average stability of a small monomeric protein is only 5 to 10 kcal/mol (after taking into account positive and negative interactions)
Shows us that evolution has resulted in only a marginal stability of the folded state over the unfolded state –> important because it allows cells to ‘remove’/degrade proteins as the folded state is only slightly energetically stable.
Outline the importance of… Covalent interactions, compaction, hierarchy, adaptability and sequence versatility (determinants of protein folding) in protein folding??
How do we determine protein stability?
Definition and causes of denaturation?
Definition Denaturation –> Loss of native structure integrity with accompanying loss of activity.
Causes of denaturation –> heat or cold; pH extremes; organic solvents or chaotropic agents: urea and guanidinium hydrochloride.
How do we plot protein unfolding curve? Are there any important point we should remember?
Plot
Denaturant - X-axis
Percent unfolded - dependent on what is being measured to gauge folding - Y axis
What is Circular Dichroism? What information about protein folding to we obtain using this analytical technique?
What are the three different types of wave oscillations? Background to CD
Why is there differential absorption in protein’s/2o structures?
How is Detla Ɛ (differential absorption calculated) for CD?
Differential absorbance (Delta epsilon) = L.H polarized light - R.H polarized light
Application: Alpha helix - more left handed light absorbed at low wavelengths resulting in positive DƐ , at around 200 there is no difference and after 200 more right handed light absorbed
Does the A.A sequence or 2o structure influence the CD absorption more at low wavelengths?
Different wavelength interaction with the A.A. and secondary structure
- A.A (small scale) more influential at smaller wavelength - A.A chirality has a greater impact on a smaller scale
- Secondary structure is more influential at larger wavelengths
Explains why at extremely large wavelengths there no distinction between 2o structures as the wavelength is too large to interact with structures.
From a measurement perspective…
What happens when equal amounts of L.H polarized and R.H polarized light are absorbed?
What happens when more R.H polarized light is absorbed relative to L.H polarized light?
Pros and Cons of using CD?
What is the Levinthal paradox?
The Levinthal paradox –> States that the folding can not be accomplished by random search/random process of folding there has to be pathway –> if it were to happen by random search - protein folding would take too long to occur too many different conformation combinations.
How is it possible to get proteins to fold in microseconds, if there are so many different possible conformations?
What is the two-state model of folding state?
No stable intermediate species between the native and the unfolded state.
What are two techniques that we can used to study protein kinetics?
- FRET –> smaller scale - individual protein basis
- Protein ensemble/Stopped flow –> allows us to examine the kinetics of the protein using a larger scale (larger samples)
What is FRET?
Fluorescence resonance energy transfer (FRET) is one example of an analytical technique that is used to examine protein folding using changes in spectroscopic signalsc –> E.g. Can be used to study kinetics of protein folding
FRET is used in order to examine folding occurring in real time on the individual protein level/molecular level.
The theory behind FRET involves…
- Attaching two fluorophores, Donor (D) and Acceptor (A), to a protein.
- Irradiating the sample, some of the fluorescence’s is absorbed by D and can potentially be transferred to A, given that they are in close proximity.
- The electronic excitation of A produces an emission that can be detected and measured.
- The degree of transfer (FRET efficiency), will depend on the distance and orientation between the two fluorophores, thus acting as a molecular ruler (Voet, Voet and Pratt, 2011).
Outline how Protein ensemble/Stopped flow can be used to examine protein kinetics?
What is the transition state theory?
How does protein compare between in-vitro and in-vivo?
Cell is a dynamic and complex environment –> this results in more factors influencing protein folding within cells
i.e. crowding, protein aggregation, cellular compartments
Main factor to consider is crowding - the cell is full of different macromolecules –> greatest source of misfolding
What is Phi (Φ) Value Analysis?
What, why and How?
How to calculate the Φ Value? How to interpret the results (Φ = 0 & 1)?
Can Φ Value Analysis can be used to identify unproductive proteins from the productive proteins that are found at local minima?
Yes, Φ Value Analysis can be used to identify unproductive proteins from the productive proteins that are found at local minima
What is Pulsed H/D exchange?
Pulsed H/D exchange (combining stopped flow and NMR) –> another way of examining protein folding in real time
Follows individual residues in a protein over time
Weakly acidic protons (amine and hydroxyl groups) exchange H+ with water in a process known as hydrogen exchange –> as hydrogen and deuterium have a different frequency range in NMR we are able to follow this exchange
Proteins in vivo have many protons that are exchangeable for deuterium (i.e. backbone amide groups) but protons involved in hydrogen bonding are not involved in this exchange plus internal residues do not participate. Hence by combining pulsed H/D exchange and NMR we can follow protein folding in real time.
Provide an overview of Pulsed H/D exchange.
In FRET analysis what donor and acceptor groups can we utilize?
In proteins, the donor and acceptor can be the side chains of Trp and Tyrosine residues (Absorb/emit in the U.V. spectrum) or alternatively we can add fluorescent molecules to reactive side chains (Cys)
This then allows us to track distances between particular residues as the protein folds.
Generally speaking, how does the cell control protein folding?
The cell has several quality control mechanisms to assist and control in vivo protein folding –> proteins exist (mostly using ATP) that ensure proper folding and to correct misfolded proteins
Examples of Proteins inolved in ensuring correct protein folding?
- HSP70 in E. Coli (reverses aggregation/denaturation + works with HSP40)
- Trigger factors (Don’t need ATP + associated with ribosome + acts early on during folding)
- Chaperonins (large multi subunit complexes + Type I in bacteria, mitochondria, chloroplasts + Type II in archaea and eukaryotes)
- Nucleoplasmins
- Protein disulphide isomerase (PDI)
- Peptyl prolyl isomerase (PPI)
General overview of ribosome - E. Coli? How many in the cell? Size? Different subunits? What are they composed of?
Outline how trigger factors aid in protein folding?
Provide a brief overview of the ribosomal translational process?
What does Chaperone DnaK do?
Chaperone DnaK –> either aid in folding directly (sufficient residues exposed for folding to take place) or transport to GroEL
What is Chaperonin type I? What is the basic structure?
What are the two conformational states of GroEL?
Outline how does Chaperonine type I attracts/binds to protein?
Outline the GroEL/GroES 2 stroke mechanism?
How many proteins use Chaperonine Type 1 in E. Coli? Can a protein undergo mutliple cycles with Chaperonine Type 1?
Is the Chaperonine Type 1 present in both eukaryotes and prokaryotes?
Voet & Voet 2 stroke mechanism?
Trans –> Cis?
Outline the 2 proposed models by which GroEL/ES aids in protein folding?
What are heat shock proteins? What are their general functions?
Outline what a Protein disulphide isomerase (PDI) is and how it works.
Outline the structure Protein disulphide isomerase? How has it evolved to perform its function? What are the important motifs to consider?
Outline the funcion of Peptidyl Prolyl Isomerase (PPI)?
Problems that may arise with the non-selective nature of Lysosomes? How does the cell solve this?
Outline how ubiquitination of a protein takes place (general mechanism)
Mechanism of ubiquitination? Ubiquitination is accomplished using E1 (Ubiquitin activating enzyme), E2 (Ubiquitin-conjugating enzyme) and E3 (Ubiquitin-protein ligase)
Two residues on ubiquitin can be used for the ubiquitination of proteins - Lysine 48 and Lysine 63.
Ubiquitin can form chains (polyubiquitin chains) ubiquitination using lys48 and 63 can be seen as a binary code (0 and 1) that forms tags that can be identified.
What two residues on ubiquitin can be ubiquitinated? What purpose do these two ubiquitin sites play?
Two residues on ubiquitin can be ubiquitinated - Lysine 48 and Lysine 63.
Ubiquitin can form chains (polyubiquitin chains) –> ubiquitination using lys48 and 63 can be seen as a binary code (0 and 1) that forms tags that can be identified.
Once a protein has been ubiquitinated what happens?
Once ubiquitinated –> protein is ready to be degraded by the 26S proteasome complex - ubiquitin creates affinity
Proteolysis takes place inside the barrel of the proteasome complex –> this allows the process to be extensive and processive while not damaging other cellular components
The proteasome does not degrade the ubiquitin molecules - they are just returned to the cell
Detail breakdown of the proteasome structure?
Breakdown of the 20S subunit in the proteasome structure?
Outline the catalytic model of the proteasome?
Explain the step by step process of the Aggresome-autophagy pathway including all the different players?
What are some examples of diseases associated with Protein misfolding?
What protein misfolding and aggregation is associated with Parkinsons?
Parkinson’s Disease –> α-synuclein is the protein associated with Parkinson’s
α-synuclein - Normal membrane αS is responsible for dopamine secretion/release in motor neurons (specifically the docking of vesicles with the membrane in the pre-synaptic neuron) - influences movement
But when amyloid fibers are created it builds up on the inside of the neuron and impairs dopamine release
α-synuclein is present in two states in the cell - disordered state or in the membrane bound state (partially helical)
Overview of α-synuclein neuronal role (attached below)
Note that the exact functions of α-synuclein are not know, these are just some of the most likely possibilities - reason why Parkinson’s is not fully understood.
Outline the different ways in which α-synuclein is associated with cellular toxcitiy?
There are many different ways that α-synuclein aggregation leads to toxicity –> influences a variety of different organelles in the neuron (lysosome, vesicles, mitochondria etc.)
It is important to note that there are different structures of synuclein that exist and that the most toxic form known as the Synuclein oligomers.
For example, they increase the permeability of the membrane (break upon plasma membrane) leading to an influx of Ca2+ leading to Ca2+ dysregulation and Ca2+ dependent cell death
Lewy bodies –> Synuclein aggregates together with membrane.
Net effect of the toxic effect is neuronal death
What are the two conformational states of Prion proteins?
PrP exists in two conformational states - PrPc which is a cellular protein and PrPSc which is known as scrapie protein (pathological form).
PrPSc is chemically identical to PrPc, stable but its self propagating and it can induce conversion of PrPc into PrPSc.
Mutations in this protein are also linked to disease.
Strain specificity –> different strains of PrPSc have different pathologies and structures
Overarching theme - Partially hydrolysed Prion protein aggregates form clusters of rod like particles similar to amyloid fibers –> resulting in neuronal death.
What is common to all amyloid fibrils structures?
Central to all amyloid fibrils - Cross Beta-spine - Beta sheets tightly packed (intersheet 10 Å) stacked on top of each other –> Fibre axis is perpendicular to the Beta-sheets
How are these sheets held together?
Steric zipper model - Two beta-sheets with side chains form a dry, tightly self-complementing steric zipper bonding the sheets together (basically highly complementary)
Each sheet forms large number of H-bonds (side chain and backbone) with neighboring sheets - These sheets are then stack for further propagation.
Outline how the Beta-sheets come together to form a amyloid fibril.
- Beta sheets - that come together via the steric zipper model to form a protofilament
Note - Strands in one sheet run anti-parallel to the mating sheet
- Filament - Two protofilament that come together
Tight gap (8.5 Å) is the dry interface whereas the big gap (15 Å) in the middle is the wet interface
Why is their a gap between the two protofilaments?
Due to the presence of a wet and dry interface
Wet Interface - Lined with water molecules
Dry interface - No water molecules except for water molecules that hydrate the carboxylate ions at the end of the peptide segments polar side chains are tightly interlocked with the same side chains from the mating sheet - hydrogen bonds are not formed but instead their shapes complement each other closely forming VDW interactions –> steric zipper –> basically exclusion of water
- Fibril - Created by the propoagation of filaments
What are intrinsically disordered proteins? Are they common? Do they perform a specific function?
Why are IDPs associated with a wide range of functions?
How does the energy landscape of IDPs compare to regular ordered proteins?
What is the quartet model?
How can the different proteins forms in the quartet model be distinguished?
What type of amino acids are commonly found in IDPs?
However, note that in the plit we can see that there is still a large degree of overlap - Not absolute characteristics.
Outline why NMR is useful for studying IDPs? What information can be gathered?
Example a protein that has both a ordered and disoreded region?
How can Circular Dichroism be used to identify and classify IDPs?
What function is suited for IDPs charateristics?
Acting as a hub in a Protein-protein interaction netwrok - can bind to mutiple partners
Important in Signalling cascade/networks
What are fuzzy complexes?
What are two analytical techniques that can be used to study protein-protein interactions?
Outline an example of an IDPs in Protein-membrane interaction.
As soon as Alpha-synuclein binds to a membrane - membrane associated*
Outline the disordered to ordered transition that takes place in spider silk
Outline the 4 main stages of protein folding (General terms)
What is the landscape theory protein folding?
Is the topography of the protein funnel smooth?
It is important to note that the topography of the funnel is not smooth –> meaning that the protein can get stuck in other local minima until it obtains sufficient thermal energy to pass the kinetic barrier so that it can continue the folding process
Does the Landscape theory suggest that protein folding is hierarchical?
Yup
