Williamson R A, Bartels H, Murphy G, Freedman R B
Research School of Biosciences, Biological Laboratory, University of Kent, Canterbury, UK.
Protein Eng. 1994 Aug;7(8):1035-40. doi: 10.1093/protein/7.8.1035.
The truncated forms of tissue inhibitor of metalloproteinase-1 and -2 (delta TIMP-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 delta TIMP-2 were studied: delta TIMP-2 T21 derived from the full-length cDNA clone isolated from a mixed-tumour library, and delta TIMP-2 A21 containing the highly conserved V18IRAK22 sequence. In all three delta TIMP proteins the tryptophan environments in the native state appeared to be similar, but substantial differences were seen in their conformational stabilities and refolding kinetics. delta TIMP-1 was approximately twice as stable as delta TIMP-2 T21 and 1.4-fold more stable than delta TIMP-2 A21. This stability difference between delta TIMP-1 and delta TIMP-2 was shown to be independent of N-linked glycosylation. delta TIMP-1 and delta TIMP-2 A21 both showed simple two-state refolding kinetics, whereas delta TIMP-2 T21 refolding was more complex and biphasic in character. These differences between delta TIMP-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.
金属蛋白酶组织抑制剂-1和-2的截短形式（δTIMP-1和-2），包含N端活性结构域，是通过内在荧光进行结构分析的理想分子，因为它们各自都含有一个单一的保守色氨酸残基。在本文中，我们描述了对它们的构象稳定性、去折叠/重折叠动力学以及独特色氨酸的环境的研究，这些研究是通过其在天然状态下的荧光特性以及暴露于外部淬灭剂丙烯酰胺来判断的。研究了两种形式的δTIMP-2：源自从混合肿瘤文库中分离出的全长cDNA克隆的δTIMP-2 T21，以及包含高度保守的V18IRAK22序列的δTIMP-2 A21。在所有三种δTIMP蛋白中，天然状态下的色氨酸环境似乎相似，但在它们的构象稳定性和重折叠动力学方面存在显著差异。δTIMP-1的稳定性约为δTIMP-2 T21的两倍，比δTIMP-2 A21高1.4倍。δTIMP-1和δTIMP-2之间的这种稳定性差异被证明与N-连接糖基化无关。δTIMP-1和δTIMP-2 A21都表现出简单的两态重折叠动力学，而δTIMP-2 T21的重折叠更复杂且具有双相性。δTIMP-2 T21和A21之间的这些差异表明，第21位残基是TIMP蛋白中的一个结构重要位点。所有三种截短分子都可被视为稳定的独立折叠结构域，非常适合进一步的结构分析。
