Module 2: V1 - V4 Flashcards
Is there empty space inside a protein? Is water excluded from inside proteins?
no, proteins are compact
yes
Where are polar side chains usually found on the protein?
exposed on the surface
Where are nonpolar side chains usually found on the protein?
buried in the core
Are disulfide bonds found buried in the core or on the surface of a protein?
buried in the core because disulfide bonds are nonpolar in an oxidised state
however under reducing conditions cysteine is exposed on the surface of the protein because it is polar in this state (however it will not form a disulfide bond)
Where is glycine and proline usually found in a protein? Why does glycine do this and under what conditions can proline do this?
where the polypeptide chain is turning around and changing direction
glycine does this because it has no side chain and is really flexible
proline does this where turns are compatible with its backbone geometry (therefore much less proline residues are found in comparison to glycine residues)
What forces drive protein folding?
electrostatic forces, van der Waals interactions, hydrogen bonds, hydrophobic interactions
Is the folded state favoured over the unfolded state?
yes
What is the folded state called?
the native state
What is the unfolded state called?
denatured
In solution, what will an unfolded polypeptide tend to do?
spontaneously fold up
What are factors that will denature proteins?
pH, temperature and denaturants
Protein folding is cooperative. What does this statement mean?
generally, if any part of a protein fold is disrupted interactions with the rest of the protein structure are disrupted and the remainder of the structure will be lost (there is no such thing as a half-folded protein)
Is protein folding/unfolding reversible?
yes, under the appropriate conditions
What must occur in order for a protein to refold correctly?
the protein must be allowed to refold first before subjected to oxidising conditions, otherwise the disulfide bonds would be formed in the wrong place resulting in a scrambled protein
Do disulfide bonds ‘direct’ folding?
no, it is actually folding that directs disulfide bond formation
What is the role of disulfide bonds in the overall stability of the protein?
increase the relative stability of the folded state over the unfolded state(s)
Do proteins always fold spontaneously? Why?
not always, conditions in the cell can make folding slow or impossible
this is because of molecular crowding
What is molecular crowding?
cells are highly concentrated solutions of proteins/nucleic acids/sugars/lipids etc. and as a result inappropriate interactions may occur with other molecules before the protein can fold
Why is misfolding likely occur in a nascent polypeptide as it comes off the ribosome?
because the polypeptide chain grows by sequential addition of amino acid residues to the C-terminal end of the chain and misfolding occurs because the sequence is not complete
How do chaperons help avoid misfolding?
chaperones assist by binding to unfolded/partially folded polypeptides and protecting them from misfolding
Where do chaperones temporarily bind?
bind to exposed hydrophobic regions preventing them from interacting with the wrong partners: inappropriate interactions
Do chaperones fold proteins?
no, they help avoid misfolding
What is the purpose of multiple cycles of binding and release carried out by chaperones?
allows the protein to fold into the correct conformation
What happens if the protein does not fold properly after multiple cycles of binding and release carried out by chaperones?
the protein will be sent off for degradation
What are favourable interactions in proteins?
electrostatic interactions, london dispersion (van de Waals interactions), hydrogen bonds and the hydrophobic effect
What are electrostatic interactions?
long-range interactions between permanently charged groups
What is an example of an electrostatic interaction?
salt bridges (ionic interactions), especially those buried in the hydrophobic environment which strongly stabilise the protein
What are van der Waals interactions?
medium-range weak attraction between all atoms which contributes significantly to the stability in the interior of the protein
How do hydrogen bonds interact in a protein?
interaction of N-H and C=O of the peptide bond leads to local regular structures such as ɑ helices and β sheets
What is the hydrophobic effect?
the release of water molecules from the structured solvation layer around the molecule as protein folds increases the net entropy
Why don’t molecules need net charge to form electrostatic interactions?
this is because dipoles can be formed as a result of charge separation across a molecule which leads to a “dipole moment” (based upon electronegativity)
When does a hydrogen bond occur?
when two electronegative atoms compete for the same hydrogen atom
What are components of the hydrogen bond?
the main component is electrostatic (dipole of D-H interacting with the partial negative charge on A)
in strong hydrogen bonds there is also a covalent component (based on transfer of electrons from A - H)
The electrostatic, hydrogen bond, and van der Waals interactions between two polar groups in aqueous environment is not particularly favourable because comparable competing interactions are possible with water. Why then do proteins fold?
proteins also contain nonpolar groups and water is a poor solvent for nonpolar groups
(nonpolar groups cannot form hydrogen bond networks and prefer to react with other nonpolar groups)
this process is driven by entropy
Why is a polypeptide chain more ordered when it folds?
when folded, less water is attached to hydrophobic regions as these regions are now joined together, leading to an increase in entropy as the number of free water molecules increases
Does disulfide bonding direct folding or does folding direct disulfide bond formation? Discuss the evidence.
folding directs disulfide bond formation
this is because in order for a protein to fold correctly the protein must be allowed to refold first before subjected to oxidising conditions, otherwise the disulfide bonds would be formed in the wrong place resulting in a scrambled protein
therefore folding directs disulfide bond formation
Ideally, protein folding is encoded in the amino acid sequence alone. Why then do cells have, and need chaperones?
this because the polypeptide chain grows by sequential addition of amino acid residues to the C-terminal end of the chain and misfolding is able to occur because the sequence is not complete
therefore, chaperones are required because they bind to unfolded/partially folded polypeptides and protect them from misfolding
Based on what you know about amino acid side chains, devise an experiment to measure the thermal stability of a protein ~150 amino acids long.
expose the protein to differing temperatures and measure the proportion of unfolded vs folded proteins found in solution for each temperature
determine if there is a relationship between an increase in temperature and an increase in proportion of proteins which are unfolded over proteins which are folded
