Novel Pattern Specific Isotopic Labeling of Aromatic Amino Acids

描述（由申请人提供）：本提案的目标是开发在其侧链中特异性结合13C的芳香氨基酸。该方法提供了孤立的1H-13C自旋系统，用于目前能力之外的大型大分子系统的溶液结构分析。新的创新被称为模式特异性芳香标记，因为同位素掺入在苯丙氨酸，酪氨酸和色氨酸侧链的特定已知位置。芳香酸侧链的不同化学位移可以在饱和转移实验中作为引发剂实现，并且很容易在分子间和分子内NOE实验中进行区分。为了开发一种广泛使用的方法，提出了以下目标。首先，将优化特定模式标记苯丙氨酸的生产，并对系统进行修改，使其能够单独制造特定模式的酪氨酸和色氨酸。其次，将对生产系统进行微小的修改，以使酪氨酸和色氨酸的模式特异性标记。第三，将模式特异性芳香标记结合到一系列重组蛋白中进行表征。将重点关注所有三种芳香残留物类型的侧链同位素置乱。这些目标的目标是制造一套广泛适用的、廉价的试剂，以探索超越当前能力的大分子复合物和膜蛋白。一种细菌系统已经被改造，可以制造和分泌大量的苯丙氨酸。以2-13C甘油为唯一碳源，C？C ?C？标记苯丙氨酸的13C掺入率为98%。通过培养基补充，高度（bbb90 %）的模式特异性标记苯丙氨酸已被纳入重组泛素中。虽然已经比生产ILV甲基前体便宜，但将测试提高产量的条件。将对一系列重组蛋白进行表征，以建立使用该方法的指导方针，包括高度过表达的泛素，高芳香含量的NusB，以及在折纸细菌中表达的大型硫氧还蛋白融合蛋白scytovirin。核磁共振光谱和质谱分析将用于确定同位素标记的特异性和效率。未来的研究包括开发其他特定的芳香标记模式和创建氘化版本的极大的大分子体系。

DESCRIPTION (provided by applicant): The objective of this proposal is to develop the aromatic amino acids that have specifically incorporated 13C in their side chains. This methodology provides isolated 1H-13C spin systems for the solution structure analysis of large macromolecular systems beyond current capabilities. The new innovation is termed Pattern Specific Aromatic Labeling since isotope incorporation is at specific, known positions in the side chains of phenylalanine, tyrosine, and tryptophan. The distinct chemical shifts of aromatic acid side chains can be implemented as initiators in saturation transfer experiments and are readily distinguished in inter- and intramolecular NOE experiments. To develop a widely usable methodology, the following aims are proposed. First, the production of pattern specifically labeled phenylalanine will be optimized and the system modified to individually make pattern specific tyrosine and tryptophan. Secondly, minor modifications to the production system will be made to make pattern specifically labeled tyrosine and tryptophan. Thirdly, the incorporation of pattern specific aromatic labels into a range of recombinant proteins will be characterized. Critical attention will be paid to side chain isotope scrambling for all three aromatic residue types. The goal of these aims is to make a widely applicable, inexpensive set of reagents to explore macromolecular complexes and membrane proteins beyond current capabilities. A bacterial system has been modified to make and secrete elevated amounts of phenylalanine. Using 2-13C glycerol as the sole carbon source, C?, C?, and C? labeled phenylalanine has been made with >98% 13C incorporation. A high degree (>90%) of pattern specific labeled phenylalanine has been incorporated into recombinant ubiquitin via media supplementation. Though already less expensive than ILV methyl precursors to produce, conditions will be tested for improved yield. A range of recombinant proteins will be characterized to establish guidelines for using the methodology, including highly overepxressing ubiquitin, high aromatic content NusB, and the large thioreredoxin fusion protein scytovirin expressed in Origami bacteria. Nuclear magnetic resonance spectroscopy and mass spectrometry analysis will be used to determine isotopic labeling specificity and efficiency. Future studies include developing other specific aromatic labeling patterns and creating deuterated versions for extremely large macromolecular systems. PUBLIC HEALTH RELEVANCE: This proposal aims at developing a new set of reagents that will allow the study of macromolecular complexes and membrane proteins beyond the current state of the art capabilities. These reagents will have an impact on many aspects of human health since they have potential uses in numerous fields, particularly for the study of large macromolecular protein complexes and membrane proteins. To demonstrate the utility of the new reagents and to establish guidelines for their implementation, the researchers will apply the newly developed, inexpensive reagents to multiple recombinant systems.