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. 2015 Mar 16;5:9129. doi: 10.1038/srep09129

Table 2. Thermal unfolding of apoflavodoxin variants: 3M (WT plus E20K/E72K/D126K) and mutants thereof.

  Global spectroscopic three-state fit DSC two-state fit
Protein variant ΔHNI (kcal/mol) TmNI (°C) ΔHIU (kcal/mol) TmIU (°C) ΔHND (kcal/mol) TmND (°C) ΔHVHa (kcal/mol) Inline graphic
3Mb 38.3 ± 2.8 50.0 ± 0.4 71.9 ± 2.4 66.7 ± 0.2 83.4 ± 0.2 67.7 ± 0.1 70.9 ± 0.3 0.85
3M/I59A 37.2 ± 1.1 57.6 ± 0.2 87.6 ± 2.4 68.0 ± 0.2 - -    
3M/E61K 40.9 ± 0.2 55.0 ± 0.1 83.6 ± 2.3 67.3 ± 0.8 - -    
3M/G87V 40.1 ± 1.2 58.8 ± 0.2 80.2 ± 0.4 68.8 ± 0.1 - -    
3M/I92A 34.2 ± 2.0 50.6 ± 3.1 65.8 ± 0.7 65.1 ± 1.6 - -    
3M/Q99A 47.9 ± 0.6 58.3 ± 0.1 81.9 ± 1.8 68.6 ± 0.1 - -    
3M/D100N 49.1 ± 3.2 68.0 ± 0.1 95.9 ± 1.7 69.7 ± 1.1 71.4 ± 0.2 69.2 ± 0.1 67.0 ± 0.3 0.94
3M/S121P 40.8 ± 0.1 56.2 ± 0.1 75.3 ± 1.1 68.6 ± 0.1 - -    
3M/D129A 55.1 ± 0.9 60.7 ± 0.3 83.4 ± 1.1 68.1 ± 0.1 - -    
3M/A142V 19.7 ± 2.1 66.0 ± 4.7 83.7 ± 2.8 68.2 ± 0.4 72.1 ± 0.2 69.4 ± 0.1 75.4 ± 0.2 1.05
3M/I92A/I59A 46.5 ± 1.8 63.9 ± 0.3 73.7 ± 2.0 69.9 ± 0.8 - -    
3M/I92A/G87V 52.4 ± 6.6 58.9 ± 1.8 37.1 ± 3.3 75.1 ± 9.5 - -    
3M/I92A/D100N 46.5 ± 2.2 63.2 ± 0.6 96.9 ± 6.2 69.3 ± 0.5 - -    
3M/I92A/A142V 43.6 ± 2.9 59.0 ± 0.8 56.5 ± 6.9 67.3 ± 1.9 - -    
3M/A142V/G87V - - - - 76.7 ± 0.1 74.4 ± 0.1 79.8 ± 0.2 1.04
3M/A142V/D100N - - - - 82.0 ± 0.1 70.4 ± 0.1 83.6 ± 0.2 1.02
3M/A142V/G87V/I92A - - - - 79.1 ± 0.1 74.9 ± 0.1 75.9 ± 0.2 0.96
3M/A142V/I92A/D100N - - - - 82.3 ± 0.1 70.6 ± 0.1 85.6 ± 0.2 1.04
3M/A142V/I92A/I59A - - - - 87.6 ± 0.1 70.3 ± 0.1 92.9 ± 0.2 1.06
3M/A142V/G87V/D100N - - - - 84.6 ± 0.1 74.9 ± 0.1 84.5 ± 0.2 1.00
WTb         84.4 ± 0.2 55.8 ± 0.1 51.5 ± 0.2 0.61

aVan't Hoff enthalpies, ΔHVH, have been determined by DSC, assuming a two-state protein unfolding mechanism, using the known equation: Inline graphic, where Tm is the mid-transition temperature, Cp,max is the maximal value of the excess molar unfolding heat capacity, and ΔHND is the calorimetric enthalpy. If the assumed two-state unfolding model is correct the calorimetric enthalpy, ΔHND, and the van't Hoff enthalpy, ΔHVH are equal (i.e. ΔHVHHND = 1.0). Otherwise, both enthalpies are significantly different.

bThe ΔHVHHND ratio of mutant 3M indicates its thermal unfolding is not two-state. The same applies to the WT protein included in this Table for comparison.