A minimum folding unit in the ankyrin repeat protein p16(INK4).
A minimum folding unit in the ankyrin repeat protein p16(INK4).
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DOI：
10.1006/jmbi.2000.3803
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发表时间：
2000-06
影响因子：
5.6
通讯作者：
Bin Zhang;Zheng‐yu Peng
中科院分区：
文献类型：
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作者：
Bin Zhang;Zheng‐yu Peng
锚蛋白重复序列是一种丰富的33个残基序列基序，形成连续的β-发夹-螺旋-环-螺旋（β（2）α（2））折叠。大多数锚蛋白重复序列蛋白由四个或更多个完整的重复序列组成，这些重复序列在相邻模块之间提供稳定的相互作用。细胞周期蛋白依赖性激酶抑制剂和肿瘤抑制因子p16（INK 4）（p16）是已知结构的最小锚蛋白重复序列蛋白之一。它由四个完整的重复加上短的N和C-末端侧翼区，在溶液中是非结构化的。初步的蛋白水解研究和预测的基础上，使用计算机算法识别自主折叠单位，我们已经确定了一个片段组成的第三和第四锚蛋白重复的p16，称为p16 C，可以折叠独立，没有其余的蛋白质。远紫外圆二色性研究表明，p16 C有一个显着的α-螺旋二级结构水平，和两个脯氨酸取代，破坏了野生型p16的α-螺旋二级结构破坏了p16 C的二级结构。p16 C的热变性是协同的和可逆的，转变的中点在30。5（+/-1）摄氏度。根据尿素诱导的变性研究，在20 ℃时p16 C的解折叠自由能估计为1.7（+/-0.3）kcal/mol。（1）H-（15）N 2D NMR研究表明p16 C中的锚蛋白重复序列可能折叠成与全长p16类似的结构。为了确定p16中的最小自主折叠单元，我们进一步将p16 C切割成两个互补的肽，每个肽含有一个锚蛋白重复。这些肽在溶液中是非结构化的。因此，p16 C是已知独立折叠的最小锚蛋白重复模块，并且一般而言，我们认为两个锚蛋白重复折叠可能是所有锚蛋白重复蛋白的最小结构单元。并进一步讨论了p16 C在蛋白质折叠和蛋白质工程中的意义。
The ankyrin repeat is an abundant, 33 residue sequence motif that forms a consecutive beta-hairpin-helix-loop-helix (beta(2)alpha(2)) fold. Most ankyrin repeat proteins consist of four or more complete repeats, which provide stabilizing interactions between adjacent modules. The cyclin-dependent kinase inhibitor and tumor suppressor p16(INK4) (p16) is one of the smallest ankyrin repeat proteins with a known structure. It consists of four complete repeats plus short N and C-terminal flanking regions that are unstructured in solution. On the basis of preliminary proteolysis studies and predictions using a computer algorithm for identifying autonomous folding units, we have identified a fragment consisting of the third and fourth ankyrin repeats of p16, called p16C, that can fold independently, without the rest of the protein. Far-UV circular dichroism studies showed that p16C has a significant level of alpha-helical secondary structure, and two proline substitutions that disrupt the alpha-helical secondary structure in wild-type p16 disrupt the secondary structure in p16C. The thermal denaturation of p16C is cooperative and reversible, with a midpoint of transition at 30. 5(+/-1) degrees C. From urea-induced denaturation studies, the free energy of unfolding for p16C was estimated to be 1.7(+/-0.3) kcal/mol at 20 degrees C. (1)H-(15)N 2D NMR studies suggest that the ankyrin repeats in p16C are likely to fold into a structure similar to that of full-length p16. In order to define the minimum autonomous folding unit in p16, we have further dissected p16C into two complementary peptides, each containing a single ankyrin repeat. These peptides are unstructured in solution. Thus, p16C is the smallest ankyrin repeat module that is known to fold independently and, in general, we believe that the two-ankyrin repeat fold could be the minimum structural unit for all ankyrin repeat proteins. We further discuss the significance of p16C in protein folding and engineering.