The thermodynamics of association and unfolding of the 205-316 C-terminal fragment of thermolysin.
The 205-316 C-terminal fragment of thermolysin has been studied by differential scanning calorimetry at pH values 2.5, 3.0, 3.5, 4.0 and 5.0 and at a constant ionic strength of 130 mM. The thermal unfolding of the fragment occurs at thermodynamic equilibrium under our experimental conditions. The effect of sample concentration at the different pH values on the calorimetric traces is consistent with a monomer-dimer equilibrium of the folded fragment, which undergoes thermal unfolding into individual fragments. Equilibrium sedimentation experiments at 10 degrees C and different pH values confirm the presence of the association equilibrium and provide the value of the dimerization constants. The global analysis of the calorimetric, heat capacity curves has been carried out by a multidimensional fitting to the model N2<-->2N<-->2U. The analysis leads to a complete thermodynamic characterization of both the association and unfolding processes of the fragment. The resulting thermodynamic functions suggest a partially unfolded structure for both the monomeric and dimeric fragment, as well as a conformational change linked to the association process. Our results are discussed in terms of the structural information currently available and compared with the energetics of unfolding of the shorter 255-316 dimeric C-terminal fragment of thermolysin (Conejero-Lara, F., De Filippis, V., Fontana, A. and Mateo, P.L. (1994) FEBS Lett. 344, 154-156). The presence of the additional 50 residues increases the relative population of the 205-316 monomeric fragment versus that of the 255-316 fragment.
Azuaga
A I
AI
Departamento de Química Física, Facultad de Ciencias, Universidad de Granada, Spain.
Conejero-Lara
F
F
Rivas
G
G
De Filippis
V
V
Fontana
A
A
Mateo
P L
PL
D002152:Calorimetry, Differential Scanning; D006863:Hydrogen-Ion Concentration; D017510:Protein Folding; D013816:Thermodynamics; D013820:Thermolysin / Q000737:chemistry*
D013820:Thermolysin