FOLDING OF BARNASE IN PARTS
FOLDING OF BARNASE IN PARTS
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DOI：
10.1021/bi00178a039
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发表时间：
1994-03-29
期刊：
影响因子：
2.9
通讯作者：
FERSHT, AR
中科院分区：
文献类型：
--
作者：
KIPPEN, AD;SANCHO, J;FERSHT, AR
蛋白质未折叠状态下的残余结构的延伸可能构成启动蛋白质折叠的关键区域。我们正在通过将barnase分成片段来寻找barnase中的此类区域。通过这种方式，我们可以搜索刚刚在局部序列内形成的区域。我们还采用可以检测低水平残留结构的方法。 在本研究中，我们检查了片段 1-22 和包含所有催化残基的大片段 (23-110)。片段1-22包含第一个α-螺旋，片段23-110包含第二个α-螺旋和天然芽孢杆菌RNA酶的β-片层结构形成残基。这些片段在缔合时快速而紧密地结合在一起，形成完全天然的复合物。圆二色性和荧光光谱研究表明，每个片段主要是无序的。然而，我们通过三氟乙醇滴定程序发现，在25℃的水中，约3%的片段1-22是螺旋状的。重要的是，我们检测到片段 23-110 对 GpUp 和 RNA 的残余催化活性以及结合 barnase 多肽抑制剂 barstar 的能力，表明该片段可以在水中形成类似天然的构象。催化活性不是由亲本酶或其他核糖核酸酶的少量污染性杂质引起的，因为该活性需要片段与barstar摩尔比为1:1才能完全抑制，并且在比使亲本酶变性所需浓度低得多的尿素浓度下，该活性丧失。大碎片的近紫外圆二色光谱中有一个非常微弱的信号。 CGAC（芽孢杆菌RNA酶的紧密结合底物类似物）的结合增强了这一点。这意味着片段中存在少量预先存在的结构，这些结构在结合 GpUp 或 barstar 后得到增强。因此，来自这项对片段的研究以及来自对完整酶的早期研究的证据表明，芽孢杆菌RNA酶可以通过独立折叠结构区域的关联来折叠。
Stretches of residual structure in the unfolded states of proteins could possibly constitute crucial regions that initiate protein folding. We are searching for such regions in barnase by dividing it into fragments. By this means, we can search for regions that just form within local sequences. We are also employing methods that can detect low levels of residual structure. In this study, we examine the fragment 1-22 and a large fragment (23-110) that contains all of the catalytic residues. Fragment 1-22 contains the first alpha-helix, and fragment 23-110 contains the second alpha-helix and beta-sheet structure-forming residues of native barnase. These fragments bind together rapidly and tightly upon association to form a fully native like complex. Studies by circular dichroism and fluorescence spectroscopy indicate that each fragment is mainly disordered. However, we find by a procedure of titration with trifluoroethanol that about 3% of fragment 1-22 is helical in water at 25-degrees-C. Importantly, we have detected residual catalytic activity in fragment 23-110 toward GpUp and RNA and the ability to bind the polypeptide inhibitor of barnase, barstar, suggesting that this fragment can form a nativelike conformation in water. The catalytic activity does not result from a small amount of contaminating impurity of parent enzyme or other ribonuclease, since the activity requires a 1:1 mole ratio of fragment to barstar for complete inhibition, and the activity is lost in much lower concentrations of urea than are required to denature the parent enzyme. There is a very weak signal in the near-UV CD spectrum of the large fragment. This is enhanced on the binding of CGAC, a tight-binding substrate analogue of barnase. This implies that there is small amount of preexisting structure in the fragment that is enhanced upon the binding of GpUp or barstar. Thus, evidence from this study on fragments, and from earlier studies on the intact enzyme, shows that barnase can fold by association of independently folded regions of structure.