Folding and stability of the active N-terminal domain of tissue inhibitor of metalloproteinases-1 and -2

The truncated forms of tissue inhibitor of metalloproteinase-1 and -2 (ΔTIMP-1 and -2), comprising the N-terminal active domain, are ideal molecules for structural analysis by intrinsic fluorescence as each contains a single conserved tryptophan residue. In this paper we describe studies on their conformational stability, unfolding/refolding kinetics and the environment of the unique tryptophan as judged by its fluorescence properties in the native state and exposure to an external quencher, acrylamide. Two forms of ΔTIMP-2 were studied: ΔTIMP-2 T21 derived from the full-length cDNA clone isolated from a mixed-tumour library, and ΔTIMP-2 A21 containing the highly conserved V18IRAK22 sequence. In all three ΔTIMP proteins the tryptophan environments in the native state appeared to be similar, but substantial differences were seen in their conformational stabilities and refolding kinetics. ΔTIMP-1 was approximately twice as stable as ΔTIMP-2 T21 and 1.4-fold more stable than ΔTIMP-2 A21. This stability difference between ΔTIMP-1 and ΔTIMP-2 was shown to be independent of N-linked glycosylation. ΔTTMP-1 and ΔTIMP-2 A21 both showed simple two-state refolding kinetics, whereas ΔTIMP-2 T21 refolding was more complex and biphasic in character. These differences between ΔTIMP-2 T21 and A21 suggest that residue 21 is a structurally important site in the TIMP protein. All three truncated molecules can be considered as stable independent folding domains ideally suited for further structural analysis.