Liu N, Deillon C, Klauser S, Gutte B, Thomas R M
Biochemisches Institut der Universität Zürich, Switzerland.
Protein Sci. 1998 May;7(5):1214-20. doi: 10.1002/pro.5560070517.
An artificial HIV enhancer-binding polypeptide has recently been dimerized by covalently linking it to the leucine zipper motif of the yeast transcriptional activator GCN4 (Liu N et al., 1997, Eur Biophys J 25:399-403). Although it seemed that the dimerization of this peptide could be best achieved by the use of the retro sequence of the leucine zipper, this approach was not implemented in the original construct. As the first step toward the synthesis of a basic region-retro leucine zipper HIV enhancer-binding fusion protein, we have now prepared the retro version of the leucine zipper (r-LZ35) and performed initial physicochemical characterization. Circular dichroism and sedimentation equilibrium studies showed that, at concentrations < 100 microM, the retro peptide was an unstructured monomer. At higher concentrations, however, the monomer was in equilibrium with a tetramer and, at 1 mM, the retro peptide was almost fully helical. N-terminal extension of the retro peptide by the tripeptide Cys-Gly-Gly resulted in a 38-residue polypeptide that could be covalently dimerized by forming a disulfide bond between two chains to give the peptide (r-LZ38)2. Even in the low micromolar concentration range peptide (r-LZ38)2 formed a stable, noncovalent, helical dimer as revealed by circular dichroism and sedimentation equilibrium in the presence and absence of guanidinium chloride. (r-LZ38)2 has been crystallized and X-ray structural analysis is under way. The disulfide-crosslinked retro-leucine zipper may lend itself to interesting protein structural studies, including protein design. The present work also highlights the structural and functional potential of retro proteins in general.
最近，一种人工合成的HIV增强子结合多肽通过与酵母转录激活因子GCN4的亮氨酸拉链基序共价连接而实现了二聚化（Liu N等人，1997年，《欧洲生物物理杂志》25:399 - 403）。尽管似乎通过使用亮氨酸拉链的反向序列能够最好地实现该多肽的二聚化，但在最初构建体中并未采用这种方法。作为合成碱性区域 - 反向亮氨酸拉链HIV增强子结合融合蛋白的第一步，我们现已制备了亮氨酸拉链的反向版本（r - LZ35）并进行了初步的物理化学表征。圆二色性和沉降平衡研究表明，在浓度<100微摩尔时，反向多肽是无结构的单体。然而，在较高浓度下，单体与四聚体处于平衡状态，在1毫摩尔时，反向多肽几乎完全呈螺旋结构。通过三肽Cys - Gly - Gly对反向多肽进行N端延伸，得到了一个38个残基的多肽，该多肽可通过在两条链之间形成二硫键而共价二聚化，得到肽（r - LZ38）2。即使在低微摩尔浓度范围内，通过圆二色性和在有无氯化胍情况下的沉降平衡表明，肽（r - LZ38）2形成了稳定的、非共价的、螺旋二聚体。（r - LZ38）2已结晶，X射线结构分析正在进行中。二硫键交联的反向亮氨酸拉链可能适用于有趣的蛋白质结构研究，包括蛋白质设计。本研究还突出了一般反向蛋白的结构和功能潜力。
