Li Hai, Wojtaszek Jessica L, Greene Lesley H
Department of Chemistry, Old Dominion University, 4541 Hampton Boulevard, Norfolk, VA 23529-0126, USA.
Biochim Biophys Acta. 2009 Apr;1794(4):583-93. doi: 10.1016/j.bbapap.2009.01.004. Epub 2009 Jan 29.
Computational and experimental studies focusing on the role of conserved amino acids for folding and stability is an active and promising area of research. To date however, only a small fraction of the potential superfamilies have been investigated. To further expand our understanding we present the results of a bioinformatics analysis of the death domain superfamily. The fold consists of a six helical bundle with a Greek-key topology. Our sequence and structural studies have identified a group of conserved hydrophobic residues and corresponding long-range interactions, which we propose are important in the formation and stabilization of the hydrophobic core and native topology. Six conserved hydrophilic residues were additionally identified and may play a functional role during apoptosis. We also report the establishment of an experimental system that will facilitate studies to test the role of the conserved residues in folding and stability. Equilibrium unfolding and refolding studies of a model superfamily member, Fas-associated death domain protein indicate that the process is cooperative, two-state and reversible. Stopped-flow fluorescence studies reveal that the folding is rapid and biphasic with the majority of the hydrophobic core forming in the first phase. Site-directed mutagenesis studies indicate that conserved tryptophans 112 and 148 are important to structure, native state stability and folding. These results present the earliest conservation analysis and biophysical characterization of the Fas-associated death domain.
专注于保守氨基酸在折叠和稳定性方面作用的计算和实验研究是一个活跃且有前景的研究领域。然而，迄今为止，仅对一小部分潜在的超家族进行了研究。为了进一步拓展我们的认识，我们展示了死亡结构域超家族的生物信息学分析结果。该折叠结构由具有希腊钥匙拓扑结构的六螺旋束组成。我们的序列和结构研究确定了一组保守的疏水残基以及相应的长程相互作用，我们认为这些在疏水核心的形成和稳定以及天然拓扑结构中很重要。另外还确定了六个保守的亲水残基，它们可能在细胞凋亡过程中发挥功能作用。我们还报告了一个实验系统的建立，这将有助于开展研究以测试保守残基在折叠和稳定性方面的作用。对模型超家族成员Fas相关死亡结构域蛋白的平衡去折叠和重折叠研究表明，该过程是协同的、两态的且可逆的。停流荧光研究揭示，折叠过程迅速且具有双相性，大部分疏水核心在第一阶段形成。定点诱变研究表明，保守的色氨酸112和148对结构、天然状态稳定性和折叠很重要。这些结果呈现了对Fas相关死亡结构域的最早的保守性分析和生物物理特性描述。
