Flashcards
the protein folding problem
Cards in this deck (27)
- 1Name the enzyme that was used in Anfinsens protein experimentsRibonuclease A
- 2what kind of experiment did anfinsen do to gain an insight into protein foldingdenaturation
- 3why did anfinsen use ribonuclease A- there was no methods of recombinant protein production
pure samples were hard to come by
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present in high abundance due to a meat processing company
Produced as a byproduct - 4describe ribonuclease A structure- Small protein, stable, contain 4 disulphides bonds within the folded protein
- 5what are the ways that a protein can be denatured- Temperature (irreversible)
- Chaotropes
- extremes of pH
- Detergents
- Mechanical stress (beaten egg whites)
- Reducing agents - 6what methods did anfinsen use to denature ribonuclease Achaotropes (urea) and reducing agents (e.g. b-mercaptoethanol)
- 7what is the effect of chaotropes (urea) on proteinsUrea is a chaotropic chemical that is able to denature chemical as it can competes with weak internal interactions that hold the protein together e.g. hydrogen bonds and polar interactions
- This molecule contains hydrogen acceptors and donors, if applied at high enough concentrations it competes with these interactions
- Generally used in high concentrations
- The same is true for guanidium chloride - 8describe the effect of SDS on proteinsSodium dodeyl sulfate (SDS)
- Commonly used to denature proteins
- E.g. SDS page
- Running buffer in SDS page breaks the hydrophobic interactions - 9describe the effects of reducing agents on proteinsbreak disuplide bonds
- 10how can protein unfolding be measured- Enzymatic assays- can be used to distinguish between folded and unfolded non functional proteins
- UV spectroscopy can also be used to measure the denaturation of a protein - 11summarise anfinsens experiment (part 1)- ribonuclease A subjected to denaturation by urea
- Addition of reducing agent- structure contained disulphide bonds. Therefore, to fully have the denatured structure of the enzyme you have to break these by reduction
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exposed the solution to oxygen= depletion of the reducing agent
Further to this Anfinsen removed the urea (dialysis?)
let it equilibrate, Anfinsen tested the enzymatic activity and discovered that hardly any activity was recovered by this - 12summarise anfinsens experiment (part 2)Anfinsen then carried out the experiment again, this time he first dialysed the sample with the presence of the urea
- After the removal of the urea, he then allowed slow oxidation of the sample
- Found that he completely recovered the activity of the RNase
- At the time of this discovery this was ground-breaking - 13what could anfisen conclude from his experimentsDisulphide bonds only form after a polypeptide has adopted a folded conformation
-All information about the native conformation of the protein is contained in the amino acid sequence - there is no other factors or information required to form the native fold of the protein other than the primary structure
- The native structure (at least in small globular proteins) is a unique, stable and kinetically accessible minimum of free energy
-But native fold is not necessarily the most stable (lowest energy) form of a protein --> when a protein folds it always fold into the native structure with the lowest free energy - 14why did anfinisens initial experiment failprotein is still unfolded due to the chaotropic reagent but the cystines are now under oxidising conditions and therefore cystine bonds form in a random manner
- Within the protein there is 8 cystines which form 4 disulphide bonds. There is 105 different combinations that can be formed when these are formed randomly-
Without the correct combination the correct structure cannot be formed- there - 15what controls the confirmation that a protein can formbond rotations in the protein backbone
- There is a limitation to the flexibility in a polypeptide
- The limitation is that there is partial double bond character
- Double bonds cannot rotate --> peptide bonds are not freely rotatable leading to there being a limitation in the structural possibilities - 16what are torsional angles- There are 2 different bond angles that can describe the rotation between different amide planes
- These are called phi and Psi
- Phi angle describes the rotation around nitrogen and alpha carbon
- Psi describes the rotation between the alpha and the amino carbon - 17what is the phi anglerotation around nitrogen and alpha carbon of the amide
- 18what is the psi bondrotation between the alpha and the amino carbon
- 19describe the mobility of peptide bondsPeptide bonds cannot rotate and are therefore stiff
- 20what should also be considered when looking at the possible phi and psi angle-side chains
rotation around the phi and psi
- If they are too close to each other this too is also physically impossible
- Only certain values of the angles is allowed - 21what is a ramachanran plothows the combinations of the phi and psi that are allowed and those that produce high energy and unfavourable states
-
- Shows where the secondary structure elements fit into this plot
- Shows the allowed combination and satify the hydrogen bond requirements of the protein strucutre - 22what residue should be given allowance in consideration of the bond angles that is can formglycine- this is due to the lack of side chains in the glycine residue and therefore there is more allowed angles due to clashes being less likely
- These are commonly found in linking regions that are only allowed by glycine - 23what is levinthals paradoxAssuming that there is only 3 possible states for every flexible dihedral angle in the backbone it would take a protein of 100 amino acids 10^29 years to sample all possible confirmations
- To put this into perspective the universe is only around 1.37 x 10^18 years old - 24How is it possible that a protein adopts a well-defined fold within a relatively short period of time? (us-s)biased search
- Thought experiment based on the hamlet phrase "Methinks it is like a weasel"
- Taking this an comparing this to a protein sequence- there is not single match to this in a sequence data base
- Showing how unlikely it is for protein to fold into compatible proteins
- Monkey theory experiment -> how long does it take a chimp to type this sequence by chance
- When the process is completely random 10^40 keystrokes, however assuming a letter is types in the correct position it will not be changed
- This is a biased search and reducing the keystrokes to >1000
- Can bring the structure from randomness - not all the possible combinations of sequence and folding have to be sampled
- Evolution can bring about structure from randomness within seconds - 25when are alpha helices formed after transcription? how would you measure thisNMR
- Alpha helices can fold within nanoseconds
- Synergistic effect of the forming of hydrogen bonds within an alpha helices
- One hydrogen bond often cause the forming of another to form quickly after
- Often alpha helices are already formed when the protein is made - 26when are beta turns formed after transcription?- Longer range interactions
- Less likely that these atoms meet
- Takes closer to a microseconds for these to form - 27when are beta sheets formed after transcription?- Takes the longest time of the secondary elements for these to form
- Takes several microseconds
- This is due to the N and the C terminus being brought in close proximity --> sequentially remote
- Bonding between distal ends of the protein in some cases, unlike the alpha helices
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- Protein structure and function Flashcards on the protein folding problem , created by Samantha bruce on 05/12/2019.
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