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 (TIM P-1) fused with an N-terminal polyhistidine tag. Emission and quenchin g studies suggested that the single tryptophan is on the protein surfa ce partially exposed to solvent. The TIMP-1 recombinant unfolded rever sibly in the presence of guanidinium chloride with the transition midp oint at 2.35M extrapolation gave a stabilization free energy of 5.1. k cal 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 compa rable 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. (C) 1998 Academ ic Press.