Folding Mechanisms of TIM Barrel Proteins

TIM 桶蛋白的折叠机制

• 批准号: 8051562
• 负责人: C Robert Matthews
• 金额: $ 35.84万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-05-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8051562
• 关键词: Algorithms; Amides; Amino Acid Sequence; Amino Acids; Architecture; Biochemical Reaction; Biochemistry; Bioinformatics; Biological Assay; Biology; Biophysics; Biotechnology; Catalysis; Circular Dichroism; Circular Dichroism Spectroscopy; Complement; Databases; Detection; Deuterium; Development; Devices; Dimensions; Elements; Energy Transfer; Event; Fluorescence Spectroscopy; Free Energy; Goals; Grant; Health; Hydrogen; Hydrogen Bonding; Industry; Isoleucine; Isotopes; Kinetics; Label; Laboratories; Length; Leucine; Life; Mass Spectrum Analysis; Measurement; Medicine; Methods; Molecular; Molecular Conformation; Monitor; Organism; Oxygen; Pathway interactions; Peptides; Physiologic pulse; Play; Process; Property; Protein Engineering; Proteins; Protocols documentation; Reaction; Recovery; Relaxation; Roentgen Rays; Role; Sequence Analysis; Shapes; Side; Specificity; Staging; Structure; Surface; System; Techniques; Technology; Testing; Thermodynamics; Time; Valine; base; design; infrared spectroscopy; insight; interest; millisecond; nano; polypeptide; protein aggregate; protein folding; research study; temperature jump; tool

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.

描述（由申请人提供）：（B/a）8 TIM桶基序是生物学中最常见的基序之一，支持来自所有三个生命超级王国的生物体中的许多生化反应的催化。已经证明了链拓扑结构在形成TIM桶的折叠自由能表面中的主要作用，氨基酸序列在指导一组同源TIM桶的天然构象的快速和有效形成中的作用现在将用各种生物物理工具来探测。X射线散射（SAXS/WAXS）和Fvrster共振能量转移（FRET）技术将评估化学变性状态下的全局，区域和成对特定尺寸，以探测可能影响折叠最早阶段的非随机结构。连接到FRET，SAXS/WAXS和圆二色性（CD）检测系统的微通道混合装置将能够对微秒折叠反应进行尺寸分析，并在折叠的早期事件中评估全局二级结构。一个合作的努力将安装研究纳米到微秒的折叠反应的稳定的bab积木，从本地TIM桶切除，温度跳跃（T-跳跃）荧光和红外光谱（IR）。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。将探讨通过应用氢交换质谱技术从脉冲猝灭氘标记的中间体的蛋白水解酶中提取的肽序列的结构的瞬态和稳定的折叠中间体的关系。来自低序列同一性的直向同源和旁系同源TIM桶的组的结果的比较将允许几种算法的稳健测试，以从序列和/或拓扑预测中间体的结构。迄今为止未被认识到的，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元素的寄存器中的作用将通过突变分析来探讨。TIM桶结构数据库中保守和非保守ba和ab发夹夹的生物信息学分析将使得能够发展假说来解释夹的子集对结构和稳定性的出乎意料的大贡献。该分析还可以使得能够预测未知结构的桶中的夹具，从而增强从序列预测结构。预计这些结果将大大增加对TIM桶蛋白折叠机制的理解，特别是氨基酸序列在指导这一过程中的作用。 公共卫生关系：所获得的见解和发展的概念应该有广泛的应用到其他图案的折叠机制，从而提高了生物学的基本过程的理解。结果也可能被证明是有用的序列的结构的预测和合理设计的协议，在生物技术工业中的错误折叠或聚集的蛋白质的回收。最后，TIM桶常见的早期错误折叠反应也可以提供致病性错误折叠反应的见解。