Santiago-Rivera Z I, Williams J S, Gorenstein D G, Andrisani O M
Department of Chemistry, School of Science, Purdue University, West Lafayette, Indiana 47907.
Protein Sci. 1993 Sep;2(9):1461-71. doi: 10.1002/pro.5560020910.
In this paper we describe the expression and purification from bacteria of the recombinant basic leucine zipper (bZip) domain of the cAMP response element binding protein, CREB327. The bZip peptide, CREB259-327, purified to near homogeneity, maintains the sequence-specific CRE site recognition demonstrated by in vitro competition assays. Alkylation of the three cysteine residues of CREB259-327 was employed to prevent aggregation of the peptide due to cysteine oxidation. The Kd of the purified native and modified CREB259-327 for the CRE site was determined by gel retardation assays to be on the order of 10(-7) M. We employed CD spectroscopy to study the folding properties of the native and modified CREB259-327. The CD analyses of the native/modified CREB259-327 peptide demonstrated a 20% increase in the alpha-helical content upon binding to the cAMP response-element. Only a 5% increase in the alpha-helical content of CREB259-327 is observed upon binding to the AP-1 site. This observation contrasts with CREB from the GCN4 protein (Weiss, M.A., et al., 1990, Nature 347, 575-578). In addition, the two-dimensional (2D) 1H-NMR studies of the bZip CREB peptide further support the distinct features of the CREB protein, in comparison to GCN4. Analysis by CD and 2D NMR of the dimerization domain of CREB suggests that the distinct DNA binding characteristics of CREB reside in the basic portion of the bZip module.
在本文中，我们描述了从细菌中表达和纯化cAMP反应元件结合蛋白CREB327的重组碱性亮氨酸拉链（bZip）结构域的方法。纯化至接近均一的bZip肽CREB259 - 327，保持了体外竞争试验所证明的序列特异性CRE位点识别能力。通过对CREB259 - 327的三个半胱氨酸残基进行烷基化，以防止肽因半胱氨酸氧化而聚集。通过凝胶阻滞试验测定纯化的天然型和修饰型CREB259 - 327与CRE位点的解离常数（Kd）约为10^(-7) M。我们采用圆二色光谱（CD光谱）研究天然型和修饰型CREB259 - 327的折叠特性。天然型/修饰型CREB259 - 327肽的CD分析表明，与cAMP反应元件结合后，α - 螺旋含量增加了20%。而与AP - 1位点结合时，CREB259 - 327的α - 螺旋含量仅增加5%。这一观察结果与来自GCN4蛋白的CREB不同（Weiss, M.A., 等人，1990年，《自然》347卷，575 - 578页）。此外，bZip CREB肽的二维（2D）1H - NMR研究进一步支持了与GCN4相比，CREB蛋白具有独特特征。通过CD和2D NMR对CREB二聚化结构域的分析表明，CREB独特的DNA结合特性存在于bZip模块的碱性部分。
