Structure of high molecular weight protein systems by solution NMR spectroscopy

前言*多核(1H，13C，15N)多维溶液核磁共振波谱是一种在原子分辨率下确定蛋白质结构的有效方法。30 kDa系统的研究受到快速信号衰减的阻碍，尽管这可以通过将感兴趣的蛋白质氢化(用2H取代1H)来部分绕过这一点。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，这是通过向生长介质中添加适当标记的氨基酸(或其前体)来实现的，然后将其合并到1H-13C标记的甲基基团中(在ALA、Ile、Leu、Met、Thr或Val中)。围绕甲基C-C轴的快速旋转极大地减弱了甲基中的信号衰减(几乎10倍)，使它们成为溶液核磁共振的理想探针。这种方法的明显问题是缺乏可获得的非甲基氨基酸残基(Cys、Asp、Glu、Phe、His、Lys、Asn、Pro、Gln、Arg、Ser、Trp和Tyr)侧链的结构信息。*1H-13C基团的快速信号衰减*排除了大系统的溶液核磁共振，甲基除外。然而，1H-12C基团很容易观察到。起源于1H-12C基团的磁化可以通过核Overhauser增强(NOE)转移到甲基1H-13C基团或主链酰胺1H-15N基团，从而在所有这些位置产生重要的结构信息。我们已经在我们的初步数据中证明了这种方法的可行性。*目标*1)利用大肠杆菌作为表达系统来生产具有最佳位置的1H-12C基团的氢化蛋白。*我们将为大肠杆菌找到最佳的碳源，以策略性地将1H-12C基团整合到其他高度氢化的蛋白质中。将开发一个模型多肽系统，利用核磁共振技术对每个氨基酸中每个原子的同位素掺入模式进行定量。这将使我们能够了解底物在中心代谢和氨基酸生物合成途径中是如何处理的。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以限制同位素干扰。*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。*将使用最佳的同位素标记策略来生产完整的膜蛋白PAgP。高分辨结构将根据本提案中引入的主链酰胺1H-15N、甲基1H-13C和1H-12C之间的NOE来确定。这将使我们能够表征PAgP的活性中心，以确定其催化机理。*影响*建议的研究具有广泛的适用性，扩大了可用溶液核磁共振研究的大系统(如膜蛋白质、多结构域蛋白质和蛋白质复合体)的范围，并极大地提高了可获得的结构的质量。

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.********