Rajan S S, Denhardt D T, Lackland H, Ludescher R D
Division of Life Sciences, Rutgers University, Piscataway, New Jersey 08855-1059, USA.
Biochem Biophys Res Commun. 1998 Jan 14;242(2):303-9. doi: 10.1006/bbrc.1997.7960.
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.
利用内源荧光检测与N端多组氨酸标签融合的小鼠金属蛋白酶组织抑制剂（TIMP-1）活性N端结构域的稳定性。发射和猝灭研究表明，单一色氨酸位于蛋白质表面，部分暴露于溶剂中。TIMP-1重组体在氯化胍存在下可逆地展开，转变中点为2.35M；外推得出25℃时的稳定自由能为5.1千卡/摩尔。对荧光强度的温度依赖性分析得到一个熔解转变，中点为51℃，展开时的焓变和热容变化分别为32千卡/摩尔和0.45千卡/（开尔文·摩尔），这些值与其他单结构域蛋白相当。与文献数据比较表明，尽管小鼠重组TIMP-1与人类TIMP-1蛋白的同源性更高，但其稳定性更类似于人类金属蛋白酶抑制剂TIMP-2而非TIMP-1。
