Structure of high molecular weight protein systems by solution NMR spectroscopy

***多核（1H, 13C, 15N）多维溶液核磁共振光谱是一种在原子分辨率上测定蛋白质结构的强大方法。bbb30 kDa系统的研究受到快速信号衰减的阻碍，尽管这可以通过对感兴趣的蛋白质进行氘化（用2H代替1H）来部分规避。然而，大多数溶液核磁共振实验需要1H原子，因此必须明智地重新引入这些原子。目前，这是通过在生长培养基中添加适当标记的氨基酸（或其前体）来实现的，然后将其掺入氘化蛋白质中的1h - 13c标记的甲基（Ala, Ile, Leu, Met， Thr或Val）中。甲基C-C轴的快速旋转极大地减弱了甲基的信号衰减（几乎是10倍），使它们成为溶液核磁共振的理想探针。该方法的明显问题是缺乏可获得的非含甲基氨基酸残基侧链（Cys、Asp、Glu、Phe、His、Lys、Asn、Pro、Gln、Arg、Ser、Trp和Tyr）的结构信息。******原理*** h - 13c基团的快速信号衰减排除了大体系的溶液核磁共振，除了甲基的情况。然而，h - 12c基团很容易观察到。源自1H-12C基团的磁化可以通过核Overhauser增强（NOE）转移到甲基1H-13C基团或主酰胺1H-15N基团上，从而在所有这些位点上获得重要的结构信息。我们已经在初步数据中证明了这种方法的可行性。******目的***1)利用大肠杆菌作为表达系统，优选h - 12c基团，制备氘化蛋白。***我们将为大肠杆菌找到最佳碳源，策略性地将h - 12c基团整合到其他高度氘化的蛋白质中。一个模型肽系统将开发定量同位素结合模式，在每个原子在每个氨基酸使用核磁共振。这将使我们了解底物是如何在中心代谢和氨基酸生物合成途径中加工的。我们将首先研究丙酮酸、鼠李糖和富马酸作为碳源，以及草酸和丙二酸作为代谢抑制剂来限制同位素混乱。******2)通过溶液核磁共振光谱确定具有挑战性的蛋白质的高分辨率结构。***将采用最优同位素标记策略生产完整膜蛋白PagP。根据本提案中引入的主酰胺1H-15N、甲基1H-13C和1H-12C位点之间的noe，确定高分辨率结构。这将使我们能够表征PagP的活性位点，以确定其催化机制。******影响***本研究具有广泛的适用性，扩大了溶液核磁共振可研究的大体系（如膜蛋白、多结构域蛋白、蛋白质复合物）的范围，大大提高了可获得结构的质量********

Introduction***Multinuclear (1H, 13C, 15N) multidimensional solution NMR spectroscopy is a powerful method for determining protein structure at atomic resolution. Studies of systems >30 kDa are hampered by rapid signal decay, though this can be partially circumvented by deuterating the protein of interest (replacing 1H with 2H). Most solution NMR experiments require 1H atoms, however, so these must be judiciously re-introduced. This is currently achieved by adding appropriately labeled amino acids (or their precursors) to the growth media, which are then incorporated into 1H-13C-labeled methyl groups (in Ala, Ile, Leu, Met, Thr, or Val) in an otherwise deuterated protein. Rapid rotation about the methyl C-C axis greatly attenuates signal decay in methyl groups (almost 10-fold), making them ideal probes for solution NMR. The obvious problem with the approach is the lack of obtainable structural information for the sidechains of non-methyl-containing amino acid residues (Cys, Asp, Glu, Phe, His, Lys, Asn, Pro, Gln, Arg, Ser, Trp, and Tyr).******Rationale***Rapid signal decay in 1H-13C groups precludes solution NMR of big systems except in the case of methyl groups. However, 1H-12C groups are readily observable. Magnetization originating on 1H-12C groups can be transferred to methyl 1H-13C groups or backbone amide 1H-15N groups through the nuclear Overhauser enhancement (NOE), yielding important structural information at all of these sites. We have demonstrated the feasibility of this approach in our preliminary data.******Objectives***1) Use E. coli as an expression system to produce deuterated protein with optimally located 1H-12C groups.***We will find optimal carbon sources for E. coli to strategically incorporate 1H-12C groups into otherwise highly deuterated proteins. A model peptide system will be developed to quantitate isotope incorporation patterns at every atom in every amino acid using NMR. This will allow us to understand how the substrates are processed in the central metabolic and amino acid biosynthetic pathways. We will initially examine pyruvate, rhamnose, and fumarate as carbon sources, along with oxalate and malonate as metabolic inhibitors to limit isotope scrambling.******2) Determine high-resolution structures of challenging proteins by solution NMR spectroscopy.***An optimal isotope labeling strategy will be used to produce the integral membrane protein, PagP. A high-resolution structure will be determined on the basis of NOEs between backbone amide 1H-15N, methyl 1H-13C, and 1H-12C sites introduced in this proposal. This will allow us to characterize the active site of PagP to determine its catalytic mechanism.******Impact***The proposed research is widely applicable, extending the range of big systems (like membrane proteins, multi-domain proteins, and protein complexes) that can be studied by solution NMR and greatly improving the quality of structures that can be obtained.********