Structural basis for the difference in thermodynamic properties between the two cysteine proteinase inhibitors human stefins A and B

Homology modelling has been used to model stefin A based onthe X-ray structure of stefin B. Several models have been producedby interactive modelling or positioning of the side chains byMonteCarlo procedure with simulated annealing.The quality of modelswas evaluated by calculation of the free energy of hydration,3D-1D potential or buried area of surface accessibility. StefinA is a thermostable protein, exhibiting a two-state denaturation,while stefin B denaturesat a 40°C lower temperature andforms a stable molten globule intermediate under mild denaturingconditions. From the tertiary structures, thermodynamic functionswere predicted, conforming closely to the experimental calorimetrkresults. Polar and apolar buried areas of surface accessibilitywere obtained by structural deconvolution of the thermograms.It is suggested that the bask difference between the stefinsis the domination of hydrophobic interaction in the stabilizationof stefin B, which is due to its non-specific nature leadingto the formation of a molten globule intermediate. Modellingof stefin A predicts increased numbers of hydrogen bonds whichstabilize it and the increase the cooperativity of its denaturation.