N rightarrow D transitions that are not two-state often give the appearance of a
ID: 1065019 • Letter: N
Question
N rightarrow D transitions that are not two-state often give the appearance of a less cooperative transition compared to a true two-state transition. Given this general observation, is the analysis of chemical denaturation data for a non-two state transition likely to overestimate or underestimate Delta G degree_H2o for N rightarrow D ? Explain. (b) What control should be performed in chemical denaturation experiments to establish whether or not a process is likely 2-state? (c) Thermal denaturation is more likely than chemical denaturation to result in aggregation of the denatured peptide. Will Delta G degree for N rightarrow D be overestimated or underestimated in differential scanning calorimetry (DSC) experiments where such aggregation occurs? Explain. (d) What control should be performed in DSC experiments to establish whether or not aggregation is interfering with the measurement of excess heat capacity?Explanation / Answer
1) Denaturation experiment
(a) For N --> D transitions occuring in two step process are often found to appear as a single state transition. Appearentce of single state instead of a true two state transition would result in a lower value that is underestimate dGo(H2O) for N --> D. the total free energy change for the process native to denaturated state is a sum total of all the free energy changes in the system. Since only on state is appearing, it would reduce the overall free energy change.
(b) We could run the N --> D transition process at different temperatures to analyze If the process is a two step or a one strp transition. At lower temperature the transition rate slows down, and we would clearly see a two step transition If existed, while at higher temperature rapid transition occurs which would give a single state only.
(c) thermal denaturation occurs more likely than chemical denaturation. This results in formation of aggregates of the denatured peptides. The free energy change for this thermal denaturation resulting in aggregation of denatured peptides would have a value higher that is would be overestimated in differential scanning calorimetry (DSC) experiment. The total heat capacity of the process calculated here would be higher which increases the free energy change of the system.
(d) One has to run the experiment under both thermal as well as chemical denaturation conditions to analyse If the aggregation is influencing the excess heat capacity measured for the given experiment. Chemical denaturation value If comes to be the same as the thermal denaturation value, than no significant effect exists due to thermal denaturation.
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