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?
What happens if proteins do not reach their native state in GroEL/ES?
Proteins that have not achieved the native state are quickly recaptured by GroEL whereas folded proteins lack the exposed hydrophobic residues.
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)?
Definition protein turnover?
Definition: Protein turnover is the balance between protein synthesis and degradation –> Cells need to be able to turn on and off proteins at different times so the cell has mechanisms of destruction i.e. Lysosome/endosome, proteasome, etc.
Importance of protein turnover?
- Eliminate abnormal proteins which when accumulates could be harmful
- Allow for the control/regulation of cellular metabolism by eliminating superfluous enzymes and regulatory proteins.
How do most protein abnormalities arise?
Most abnormal proteins arise due to chemical modification/spontaneous denaturation in the reactive cell environment rather than mutations or rare errors in transcription/translation.
What protein are the Endosome-lysosome pathway & Ubiquitin-proteasome pathway responsible for?
Endosome-lysosome pathway degrades extracellular and cell-surface proteins
Ubiquitin-proteasome pathway degrades proteins from the cytoplasm, nucleus and ER.
Normally speaking, does the rate of protein degradation, independent of catalysis, fluctuate?
Yes
Depending on the type of protein, the degradation takes place at different rates
- Ornithine decarboxylase has a half-life of 11 minutes whereas
- Gamma-Crystallin (eye lens protein) lasts as long as the organism does –> length could depend on function (catalytic or structural).
What two systems do eurkayotes use for protein degradation?
Lysosomal mechanisms
ATP driven-dependent cytosolically based mechanisms –>
Outline how the Lysosomal Protein degradation pathway works?
Lysosomal Protein degradation –> Lysosomes are cellular vesicles with an acidic pH (5.5 – maintained by proton pump ATPase) containing proteolytic enzymes (e.g., papain-like cysteine protease, serine proteases, aspartic proteinases, etc.) used for the breakdown of proteins
Highly active at an acidic pH but not cytosolic pH –> protect against leakage.
Lysosomes degrade intracellular proteins by fusing with membrane-enclosed autophagic vacuoles and similarly breaking down extracellular proteins that have been taking in by endocytosis.
Lysosomes mostly degrade proteins non-selectively –> evident by the fact that lysosomal inhibitors don’t affect the rapid degradation of abnormal or short-lived enzymes.
Problems that may arise with the non-selective nature of Lysosomes? How does the cell solve this?
How are proteins tagged for proteasome degradation/ATP-dependent protein degradation?
Proteins that are linked to ubiquitin (small protein 76 A.A.) via a isopeptide bond are tagged for degradation
Hence, ubiquitination tags a protein for proteasome degradation.
Where on the protein does ubiquitination take place?
It can take place on the C-term of Gly76 - Side chain ubiquitination also occurs using lysine residues
Note that multiple ubiquitin molecules can be attached - acts like a barcode (known as a polyubiquitin chain).
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
Products released from the proteasome complex?
Yields oligopeptides 4 to 25 residues long averaging 7 to 9 residues which can then be broken down to single amino acids by exopeptidases in the cytoplasm.
What is the proteasome? What is it’s general structure?
Proteasome –> Protein complex inside all eukaryotes (in nucleus and cytoplasm) and archaea, and in some bacteria
Function: degrades proteins to form peptides of about seven to eight amino acids long.
General Structure
Proteasome consists of…
20S proteasome (barrel shaped catalytic core) and a 19S caps which associate with the ends of the 20S proteasome
20S proteasome hydrolyzes unfolded proteins in an ATP-independent manner whereas the 19S caps function to identify and unfold the proteins.
Detail breakdown of the proteasome structure?
Breakdown of the 20S subunit in the proteasome structure?
Outline the catalytic model of the proteasome?
What do we know about the 19S subunit and it’s interaction with the 20S proteasome subunit?
The 20S proteasome requires the 19S caps to recognize ubiquitinated proteins, unfold them and feed them into the catalytic active barrel –> all of which requires ATP
Not much is known about the 19S Caps as they have a low intrinsic stability –> 9 subunits in base complex of the Cap - 6 of which are ATPase that form a ring which is in contact with the alpha ring of the 20S proteasome + cross-linking experiments show that they interact with the polyUb signal.
There is another additional 8 subunits that form the lid-complex.
What role does the 11S activator protein play in the proteasome degradation of higher Eukaryotes?
Higher Eukaryotes have an 11S activator protein that can open the channel of the 20S proteasome to polypeptides (not folded proteins)
C-terminal tails of the activator insert into pockets on the 20S proteasome’s α subunits in a way that induces conformational changes in its N-terminal tails that clear the 20S proteasome’s otherwise blocked central aperture.
What is the Aggresome-autophagy pathway?
Aggresome-autophagy pathway - crucial cellular defense system against toxic build-up of misfolded proteins
Specialized type of induced autophagy that mediates selective clearance of misfolded and aggregated proteins under the conditions of proteotoxic stress –> Basically if everything fucks up and muchos proteins are misfolded or aggregated this pathway is utilized.
What are the sequence of events that lead to use of the Aggresome-autophagy pathway?
Normally what happens when a protein is misfolded?
Protein misfolded due to genetic reasons or oxidative damage –> (1). Misfolded proteins may be refolded by chaperones (2) or tagged with Lys48-linked polyubiquitin chains for degradation by the proteasome.
What happens if the proteasomes and chaperones fail/too overwhelmed with work?
Proteins form oligomers and aggregates that can cause cytotoxicity
Solution? Aggresome-autophagy pathway.
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?
Overview of Alzheimer’s disease?
Alzheimer’s disease (most common amyloid disease) - 65+ - 10% whereas 85+ 50% –> characterized by brain tissue containing abundant surrounded by dead/dying neurons as well as cell bodies containing neurofibrillary tangles (roughly 20nm in diameter) –> leads to mental deterioration
Why does the incidence increase as we age?
Chaperons/degradation becomes less efficient which leads to aggregate formation.
What are the plaques in Alzheimer’s disease formed of?
The Plaques are formed from…
- Amyloid fibrils/fragments of 40-42 amino acids amyloid-Beta peptide which is derived from a larger receptor protein - amyloid-Beta precursor protein (APP) which is broken down sequentially by a membrane anchored enzymes known as Beta & Gamma secretase
- Hyperphosphorylated form of a protein called Tau which is normally associated with microtubules
Risk factors that may contribute to Alzheimer’s disease?
- Mutations in the amyloid-Beta precursor protein in the amyloid-Beta peptide region result in the onset of AD as it leads to a potential increase in the rate of amyloid-Beta peptide formation
Problem for people suffering from Down Syndrome as the precursor protein gene is located on chromosome 21, hence the extra chromosome 21 results in increased production of the amyloid-Beta precursor protein.
- Beta-Secretase (BACE-1) rate limiting enzyme in the formation of Amyloid peptides –> increase in BACE-1 expresion - i.e. increased BACE-AS (anti-sense) RNA protects BACE-1 mRNA transcript from degradation by blocking it form miRNA family 27
- The cholesterol transport protein (apoE) Allele ApoE4 is a major risk factor for AD thought that ApoE facilitates the aggregates of the amyloid fibrils.
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 Prion Diseases?
Prion Diseases –> Scarpie, Bovine Spongiform Encephalopathy (BSE) and Creutzfeld Jakob disease (CJD)
Collectively known as transmissible spongiform encephalopathies (TSEs) - neurons develop large vacuoles give the brain a sponge like appearance.
Prion protein (PrP) –> 280 A.A., membrane anchored cell surface signal receptor, widespread in mammals, no known function.
Scarpie protien - infectious protein go into other cells and propagate –> Spread from one cell to another.
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 are the differences between the two Prp (Prion proteins)
PrPSc is a stable conformational variant of PrPC
PrPSc –> Amino acid sequence resembles the gene sequence - ruling out post-translational modifications are source of disease + Mass spectrometry shows us that they are chemically identical
But….
CD shows that they differ in secondary and tertiary structure…
Content of alpha helices and Beta-sheet differ - plausible model suggests proposes that the N-terminal region folds to form a Beta-helix (left handed helix with 3 beta-sheets)
The higher beta sheet content would favour the formation of PrPSc as amyloid fibres
This change in conformation appears to be autocatalytic - PrPSc converts PrPC into PrPSc.
Can PrpC and PrpSc both be degraded proteolytically?
PrPSc is deposited in vesicles rather than the cell surface membrane like PrPC.
Both forms are susceptible to proteolytic degradation but…
PrPSc only loses its N-terminal leaving a resistant core known as PrP 27-30 behind which still exhibits high Beta-sheet content - this can then aggregate to form amyloid plaques
Are amyloid fibres only toxic, do they play any functional role?
No they are not only toxic!
Amyloid fibers also have roles in nature - having a function –> extremely strong structure.
What are the three main types of amyloid species?
- Amyloidogenic precursors protein (protein monomers) - transient and elusive –> normally not toxic - are important to understand the origin and as therapeutic targets
- Amyloid Fibrils are stable and highly resistant to detergents and unfolding agents - not the most toxic but they do play a role in cellular death and damage.
- Pre-fibrilar oligomers are the most toxic species - small enough to move around and interact with other cellular structures/components –> their action is subject to the ability to penetrate the cellular membrane
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?
What type of disease are IDP associated with?
Associated with neurodegenerative diseases/cancer E.g. alpha-synuclein in Parkinson
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.
Why are Proline’s common in IDPs?
Proline is a secondary structure breaker - don’t have the amide –> interferes with hydrogen bonding (no hydrogen for H-bonding) - promoting disorder.
Furthermore, poly-prolines doesn’t have the hydrogen bond so they only form transiently
Popular 2o structure are poly-prolines - pseudo-helix –> every 3 residues you return back to the same residue meaning 1 turn = 3 residues - used as a common motif for protein-protein interactions.
What analytical techniques can be used to study IDPs?
Structural approaches to study IDPs
- X-Ray crystallography very limited for IDPs as they are virtually impossible to crystallize and even if we were to crystallize it, it would represent one of the infinite conformations that the protein can adopt.
- NMR is not hindered by multiple conformations of IDPs thus making it a more viable option.
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
Why would cells have naturally unfolded proteins?
Globular proteins would have to be 2-3 times larger than their disordered counterparts in order to provide the same intramolecular interface - this provides genetic economy and reduces crowding
Furthermore, disordered regions may aid in the transport across membranes and in selective protein degradation.
Functions of natively disordered proteins?
Functions of natively disordered proteins?
Most common is binding to DNA sequences which aid in transcription, DNA repair, transposition and replication.
Other functions include intracellular signal transduction, forming phosphorylation sites and aiding other proteins/RNAs fold into their native state.
Functions of natively disordered proteins?
When binding to another molecule - IDP tends to fold into temporary secondary or tertiary structures
Outline the 4 main stages of protein folding (General terms)
What is the landscape theory protein folding?
Do proteins always take the same path down the funnel in the landscape theory of protein folding?
If we were examining a collection of unfolded polypeptides with different conformations they would all have a different position in the funnel meaning that they can’t fold via the same pathway to reach the native state
Meaning there is no single pathway or closely related pathways that a polypeptide must follow to reach the native conformation.
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
What does the Landscape theory of protein folding say abou transition states?
Local energy maxima (transition states) that govern protein folding kinetics are not specific structures as the classical theory suggests rather a group of structures.
Does the Landscape theory suggest that protein folding is hierarchical?
Yup
