Schulenburg Cindy, Löw Christian, Weininger Ulrich, Mrestani-Klaus Carmen, Hofmann Hagen, Balbach Jochen, Ulbrich-Hofmann Renate, Arnold Ulrich
Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany.
Biochemistry. 2009 Sep 8;48(35):8449-57. doi: 10.1021/bi900596j.
A promising approach to unravel the relationship between sequence information, tertiary structure, and folding mechanism of proteins is the analysis of the folding behavior of proteins with low sequence identity but comparable tertiary structures. Ribonuclease A (RNase A) and its homologues, forming the RNase A superfamily, provide an excellent model system for respective studies. RNase A has been used extensively as a model protein for folding studies. However, little is known about the folding of homologous RNases. Here, we analyze the folding pathway of onconase, a homologous protein from the Northern leopard frog with great potential as a tumor therapeutic, by high-resolution techniques. Although onconase and RNase A significantly differ in the primary structure (28% sequence identity) and in thermodynamic stability (DeltaDeltaG = 20 kJ mol(-1)), both enzymes possess very similar tertiary structures. The present folding studies on onconase by rapid mixing techniques in combination with fluorescence and NMR spectroscopy allow the structural assignment of the three kinetic phases observed in stopped-flow fluorescence spectroscopy. After a slow peptidyl-prolyl cis-to-trans isomerization reaction in the unfolded state, ONC folds via an on-pathway intermediate to the native state. By quenched-flow hydrogen/deuterium exchange experiments coupled with 2D NMR spectroscopy, 31 amino acid residues were identified to be involved in the structure formation of the intermediate. Twelve of these residues are identical in the RNase A sequence, which is a significantly higher percentage (39%) than the overall 28% sequence identity. Moreover, the structure of this intermediate closely resembles two of the intermediates that occur early during the refolding of RNase A. Obviously, in spite of considerable differences in their amino acid sequence the initial folding events of both proteins are comparable, guided by a limited number of conserved residues.
解析蛋白质序列信息、三级结构和折叠机制之间关系的一种有前景的方法是分析具有低序列同一性但三级结构相似的蛋白质的折叠行为。核糖核酸酶A（RNase A）及其同系物构成了RNase A超家族，为相关研究提供了一个出色的模型系统。RNase A已被广泛用作折叠研究的模型蛋白。然而，对于同源核糖核酸酶的折叠情况却知之甚少。在此，我们通过高分辨率技术分析了豹蛙抗癌酶（onconase）的折叠途径，它是一种来自北美豹蛙的同源蛋白，具有巨大的肿瘤治疗潜力。尽管豹蛙抗癌酶和RNase A在一级结构上有显著差异（序列同一性为28%）且在热力学稳定性上也不同（ΔΔG = 20 kJ mol⁻¹），但这两种酶具有非常相似的三级结构。目前通过快速混合技术结合荧光和核磁共振光谱对豹蛙抗癌酶进行的折叠研究，能够对停流荧光光谱中观察到的三个动力学阶段进行结构归属。在未折叠状态下发生缓慢的肽基 - 脯氨酰顺反异构化反应后，豹蛙抗癌酶通过一个途径上的中间体折叠成天然状态。通过淬灭流氢/氘交换实验结合二维核磁共振光谱，确定有31个氨基酸残基参与中间体的结构形成。其中12个残基在RNase A序列中是相同的，这一比例（39%）明显高于整体28%的序列同一性。此外，这个中间体的结构与RNase A重折叠早期出现的两个中间体非常相似。显然，尽管它们的氨基酸序列存在相当大的差异，但这两种蛋白质的初始折叠事件具有可比性，由有限数量的保守残基引导。
