Flashcards
proteins
Flashcards do conjunto (23)
- 1How does the entropy of water change on folding the proteinIn native state of protein, hydrophobic side chains are shielded from water, in denatured state hydrophobic areas are exposed and water molecules stack around them in icebergs to maximise their bonding to themselves - lowers the entropy of water and also lowers enthalpy as more h bonds are formed. The water molecules are released as the protein folds and results in a gain in entropy of the water which compensates the loss of entropy of the protein
- 2How does water effect the specific heat capacity ?Icebergs form around hydrophobic groups, as the temperature is raised the icebergs melt and their latent heat of fusion contributes to cp
- 3What is delta cp of folding and why does it become lower as the temperature is raised?12 kcal/deg/mol. Because lower because the ice bergs melt
- 4How does the enthalpy of the unfolded state change with temperature?It increases as the icebergs melt so there are fewer H bonds
- 5How does the entropy of unfolding change with temperature?Entropy increases i.e. favours the unfolded state as fewer water molecules are tied down in the icebergs that surround the denatured state
- 6Why does cold unfolding occur ?give equation for delta H - shows that as temperature decreases, delta H decreases
- 7How can you increase the temperature of cold denaturation?Add denaturant - increases delta cp, decreases tm and delta H(D-N)
- 8Describe and explain the shape of melting curve obtained from differential scanning calorimetry. Where is the tm?.How can you use this method to find delta H?heat capacity of folded is lower than unfolded, large uptake of heat in the transition between the two as the process is highly endothermic. The tm is at the maximum of the peak. The area under the peak, after you subtract the baseline is the enthalpy of unfolding.
- 9Why is melting temperature not a good indicator of the stability of a protein?deltaG = 0 therefore, tm = deltaH/Tdelta S - its just the ratio of its enthalpy and entropy of denaturation
- 10How delta H change with pH? How can you calculate delta cp from delta H?- proteins become less table with decreasing pH and the Tm becomes progressively lower - the decrease in stability results from the protonation of the carboxylic acid groups which have small enthalpies of ionisation
-Delta H decreases with decreasing pH
-Plot delta H against tm and the slope is delta Cp - 11Why do denaturants unfold proteins?- Denaturants solubilise all constituents of the protein, including its polypeptide backbone and it hydrophobic side chains, but because the denatured state is more exposed to the solvent it is preferentially stabilised
-The denaturant can stabilise groups indirectly by altering the solvent properties of water or by directly interacting with the groups of the protein - 12What does a low value of m indicate?- if it is low compared to the calculated value, it indicates that the protein does not b become highly unfolded on denaturation it may also indicate that the process is occurring stepwise rather than in single cooperative transition
- 13Why is there difficulty in predicting delta Gh2o from the equation Dg = DG(h2o) - m[D]The correlation of Dg and [D] is only linear over a small concentration range in the middle - thus going down to [D] = 0 involves a long linear extrapolation - thus calorimetry can be better
- 14Why does changing the pH denature proteins?- Primary reason is due to the fact that proteins usually have buried groups that have highly perturbed pkas e.g. glutamate pka = 4.5 in denatured state, but 1.5 in the native state, at pH7 both forms are ionised, at 4.5 the denatured state is 50% protonated and this displaces the denaturation equilibrium by a factor of 2 toward denaturation
-Also at V high or low pH you get charge build up - electrostatic repulsion - 15Explain with a diagram how apparent twi state transitions may be seen- Intermediate is more compact than denatured protein, meaning mD-N>mI-N - means intermediate is highest in energy during the transition region
- 16Why is find DDG values better from m[D]50 than finding the individual DG(h20)The presence of denaturant at concentration of [D]50 changes the overall free energy of folding by only around 10 kcal/mol which averages 0.1 kcal/mol per residue which is within the experimental error.
The value of mD-N changes only insignificantly on mutation of proteins that unfold extensively in denaturant
The equation works even when there is curvature in the plots for DG against [D] and there is good agreement between the DDG ( d50) values and the DDG values determined from calorimetry. However, high concentrations of gdmcl can mask electrostatic interactions because of its ionic strength - 17Draw a graph to show how DDG of mutation of alanine to glycine changes with the change in solvent accessible surface areaLinear, with -ve gradient - at top where change in SA is small, Ala is surrounded by exposed Hphobic surface area so mutation to gly is destabilising, at bottom right, Ala is highly solvent exposed - little surface areas is buried on folding so mutation is hardly destabilising. The linearity of the plot suggests that a Hphobic bond is operating
- 18What effect is seen with double salt bridges?Synergy as there is sharing of the entropy cost between the different interactions
- 19How does changing the ability of a helix change the stability of the protein?If we make a mutation in the face of a helix that is exposed to solvent, and the mutation does not affect interaction elsewhere in the protein, the stability of the protein increases by around the same amount as that of the helix - but this rule breaks down if we over stabilise the helix to the point where it is present in the denatured state as any further stabilisation then increases the stability of the native and denatured state equally
- 20By how much does stability decrease on deletion of a methylene group from a hydrophobic core?up to 2.5 kcal/mol
- 21How is packing density, nc, defined?The number of methyl or methylene groups in a a sphere with a 6A radius from each methylene group
- 22Why do disulphide cross links stabilise proteins? By how much?They destabilise the denatured state by reducing its conformational freedom - but only if stereochemistry is right, otherwise destabilisation can occur.
Stabilisaiton of 4 kcal/mol - 23How do you calculate DDG(stat) - of a mutation of an amino acid at position AA relative to alanine-RTln(of pAA/ pALA
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- Chemistry FlashCards sobre proteins , criado por Jenna Robinson em 12-04-2018.
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- Jenna Robinson
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