Analysis of the conformational stability of the active domain of recombinant mouse TIMP-1 by intrinsic fluorescence.

Intrinsic fluorescence was used to examine the stability of an active, N-terminal domain of mouse tissue inhibitor of metalloproteinase (TIMP-1) fused with an N-terminal polyhistidine tag. Emission and quenching studies suggested that the single tryptophan is on the protein surface partially exposed to solvent. The TIMP-1 recombinant unfolded reversibly in the presence of guanidinium chloride with the transition midpoint at 2.35M; extrapolation gave a stabilization free energy of 5.1 kcal mol-1 at 25 degrees C. Analysis of the temperature dependence of the fluorescence intensity gave a melting transition with midpoint at 51 degrees C and an enthalpy and heat capacity change on unfolding of 32 kcal mol-1 and 0.45 kcal K-1 mol-1, respectively, values comparable to other single domain proteins. Comparison with literature data indicated that the stability of mouse recombinant TIMP-1 more closely resembled that of human metalloproteinase inhibitor TIMP-2 than TIMP-1 despite closer homology to the human TIMP-1 protein.