Folding Mechanisms of TIM Barrel Proteins

描述(申请人提供)：(b/a)8 TIM桶主题是生物学中最常见的主题之一，支持来自所有三个超级生命王国的生物体中的一系列生化反应的催化。在证明了链拓扑在形成TIM桶的折叠自由能表面中的主要作用之后，氨基酸序列在指导一组同源TIM桶的自然构象快速而有效地形成方面的作用现在将用各种生物物理工具来探索。X射线散射(SAXS/WAXS)和FRET共振能量转移(FRET)技术将评估化学变性状态下的全局、区域和成对的特定维度，以探索可能影响折叠早期阶段的非随机结构。与FRET、SAXS/WAXS和圆二色(CD)检测系统接口的微通道混合设备将能够对微秒级折叠反应进行维度分析，并在折叠的早期事件中评估全球二级结构。将开展一项合作工作，通过温度跳跃(T-JUMP)荧光和红外光谱(IR)研究从本地TIM桶中提取的稳定BAB积木的纳秒到微秒折叠反应。对模块中选择性质量标记的羰基进行同位素编辑的T-JUMP IR研究将测试协同折叠反应与顺序折叠反应。利用氢交换质谱学技术，对从脉冲猝灭氘标记中间体蛋白水解液中提取的多肽，探索序列与瞬时和稳定折叠中间体结构的关系。比较来自低序列同一性的同源和同源TIM桶集合的结果将允许对从序列和/或拓扑预测中间体结构的几种算法进行稳健测试。我们将通过突变分析来探讨迄今未被认识但非常常见的侧链-主链氢键相互作用在稳定ba和ab发夹中的作用，从而在建立b和a元素的注册中的作用。在TIM枪管结构数据库中对保守和非保守的ba和ab发夹的生物信息学分析将使假设的发展成为可能，以解释钳子子集对结构和稳定性的出乎意料的大贡献。这种分析还可以预测未知结构的桶中的夹子，从而增强从序列中预测结构。这些结果有望大大增加对Tim Barrel蛋白质折叠机制的理解，特别是对氨基酸序列在指导这一过程中的作用的理解。公共卫生相关性：获得的见解和发展的概念应该广泛应用于其他基序的折叠机制，从而增强对生物学基本过程的理解。这些结果也可能被证明对从序列中预测结构和合理设计生物技术行业中错误折叠或聚集的蛋白质回收方案有用。最后，TIM桶常见的早期错误折叠反应也可能为致病错误折叠反应提供见解。

DESCRIPTION (provided by applicant): The (b/a)8 TIM barrel motif is one of the most common in biology, supporting the catalysis of a host of biochemical reactions in organisms from all three super-kingdoms of life. Having demonstrated a primary role for chain topology in shaping the folding free energy surface of TIM barrels, the role of the amino acid sequence in guiding the rapid and efficient formation of the native conformation for a set of homologous TIM barrels will now be probed with a variety of biophysical tools. X-ray scattering (SAXS/WAXS) and Fvrster resonance energy transfer (FRET) techniques will assess global, regional and pair-wise specific dimensions in chemically-denatured states to probe for non-random structure that might influence the earliest stages of folding. Microchannel mixing devices interfaced to FRET, SAXS/WAXS and circular dichroism (CD) detection systems will enable a dimensional analysis of microsecond folding reactions and the assessment of global secondary structure during the early events in folding. A collaborative effort will be mounted to study the nano- to microsecond folding reactions of stable bab building blocks, excised from native TIM barrels, by temperature-jump (T-jump) fluorescence and infrared spectroscopy (IR). Isotope-edited T-jump IR studies on selectively mass-labeled carbonyls in the modules will test for concerted vs. sequential folding reactions. The relationship of the sequence to the structures of transient and stable folding intermediates will be explored by applying hydrogen exchange mass spectrometric techniques on peptides extracted from proteolytic digests of pulse-quench deuterium-labeled intermediates. Comparisons of the results from sets of orthologous and paralogous TIM barrels of low sequence identity will allow robust tests of several algorithms to predict the structures of the intermediates from the sequence and/or topology. The role of hither-to-fore unrecognized but very common side chain-main chain hydrogen bonding interactions in stabilizing ba and ab hairpins and, thereby, in establishing the register of the b and a elements will be probed by mutational analysis. Bio- informatics analysis of conserved and non-conserved ba and ab hairpin clamps in a database of TIM barrel structures will enable the development of hypotheses to explain the unexpectedly large contributions of a sub- set of clamps to structure and stability. This analysis may also enable the prediction of clamps in barrels of unknown structure and, thereby, enhance the prediction of structure from sequence. The results are expected to substantially increase the understanding of the mechanism by which TIM barrel proteins fold and, especially, the role of the amino acid sequence in directing this process. PUBLIC HEALTH RELEVANCE: The insights obtained and concepts developed should have wide application to the folding mechanisms of other motifs, thereby enhancing the understanding of a fundamental process in biology. The results may also prove to be useful for the prediction of structures from sequences and for the rational design of protocols for the recovery of misfolded or aggregated proteins in the biotechnology industry. Finally, the early misfolding reactions common to TIM barrels may also provide insights into pathogenic misfolding reactions.