A Cost‐Effective Labeling Strategy for the NMR Study of Large Proteins: Selective 15N‐Labeling of the Tryptophan Side Chains of Prolyl Oligopeptidase
A Cost‐Effective Labeling Strategy for the NMR Study of Large Proteins: Selective 15N‐Labeling of the Tryptophan Side Chains of Prolyl Oligopeptidase
复制标题
用于大蛋白 NMR 研究的经济有效的标记策略：脯氨酰寡肽酶色氨酸侧链的选择性 15N 标记
DOI：
10.1002/cbic.200900575
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发表时间：
2009
期刊：
影响因子：
3.2
通讯作者：
E. Giralt
中科院分区：
文献类型：
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作者：
T. Tarragó;Birgit Claasen;Nessim Kichik;R. Rodríguez;M. Gairí;E. Giralt
核磁共振波谱是研究蛋白质结构、蛋白质动力学和分子识别过程的有用工具，包括蛋白质-蛋白质和蛋白质-配体相互作用。[4]然而，将核磁共振实验应用于大型蛋白质仍然是一个挑战。横向弛豫过程随着大分子尺寸的增大而加速，可能需要低翻滚速率的蛋白质的氘化。因此，细胞必须在D2O，[5]中生长，这通常会降低蛋白质表达水平，并显著提高NMR样品的成本。此外，频谱的分配受到信号重叠的限制，因此通常需要通过适当的选择性标记方案来简化频谱。6]通过使用营养不良细胞株并将具有合适同位素标记的氨基酸添加到培养基中，可以实现对特定氨基酸的选择性标记。然而，考虑到氨基酸的生物合成途径是复杂的，当这些途径之一被破坏时，细胞生长可能受到限制，从而导致较低的表达水平。一种侵入性较低的方法是利用细胞的代谢机制来产生选择性标记的蛋白质，并通过对前体的特殊选择来决定一个亚群或一个特定的氨基酸最终是否被标记。许多作者描述了这样的标签方法。例如，如果将c标记的a-酮丁酸盐和a-酮异戊酸盐添加到生长培养基中，细胞随后将c标记物整合到缬氨酸、亮氨酸和异亮氨酸侧链中。同样，在培养基中加入[2-C]或[4-C]标记的吲哚，可以标记色氨酸残基。色氨酸、酪氨酸和精氨酸是特定蛋白质热点中最常见的氨基酸，因此参与了蛋白质-蛋白质和蛋白质-配体相互作用中的大部分结合能。一种适用于大蛋白质的廉价而可靠的上述残基标记策略将是非常有用的。在这里，我们报告了一种具有成本效益的标记策略，用于大蛋白质的核磁共振研究。在这种方法中，n -标签选择性地结合到蛋白质的色氨酸侧链中，无需氘化就可以获得光谱。我们将这种标记应用于脯氨酸寡肽酶（POP; EC 3.4.21.26），这是一种80 kDa的丝氨酸蛋白酶。近年来，POP已成为治疗认知障碍的重要靶点。目前正在使用一系列策略来识别POP抑制剂，因为这些化合物在实验动物中显示出神经保护和认知增强作用。从猪肌肉中提取的POP的x射线结构显示出独特的双结构域：一个具有a/b水解酶折叠的催化结构域和一个不寻常的b-螺旋桨结构域。在典型的POP共价抑制剂z -脯氨酸（Z-prolyl-prolinal， ZPP）存在时，POP的结构表明，该酶与含脯氨酸抑制剂的结合特异性是由POP色氨酸595 （Trp595）与ZPP脯氨酸环之间的疏水相互作用提供的。[16]对均匀N标记的渗透氘化POP样品（U-[H,N]-POP；图1 a）的光谱获取表明，即使使用渗透氘化和横向弛豫优化光谱（TROSY），在80 kDa的大蛋白的情况下，信号重叠仍然是一个相当大的障碍。因此，通过在最小生长培养基中添加n -吲哚，可以在色氨酸侧链上选择性标记POP样品（Trp[n -吲哚]-POP）。用质谱法检查标签的掺入情况（支持信息中的图S1）。用Trp [NIndole]-POP样品记录的[H，N]-TROSY HSQC谱显示了令人满意的信噪比（S/N），并且12个预期色氨酸信号中有11个存在并且分散良好（图1b， 2）。值得注意的是，尽管POP的分子量很高，但这一结果是在非氘化蛋白质样品中获得的。还评估了先前描述的Trp[2- c -吲哚标记的应用。然而，在渗透Trp[2-C-吲哚]标记的POP样品获得的[H，C]-芳香TROSY光谱中，仅检测到少数信噪比较差的信号（见SI）。因此，比较选择性Trp[2- c -吲哚]和Trp[n -吲哚]标记的POP结果表明，Trp[n -吲哚]标记在大蛋白质的情况下更方便。这可以通过考虑a . T. Tarrag博士，B. Claasen博士，N. kikik博士，E. Giralt教授在生物医学研究所，Baldiri Reixac, 10,08028巴塞罗那（西班牙）传真：（+ 34）93-4037126电子邮件：ernest.giralt@irbbarcelona.org [b]巴塞罗那大学有机化学系E. Giralt教授，巴塞罗那（西班牙）[c]哈佛医学院生物化学和分子药理学博士R. A. Rodriguez-Mias, 240 Longwood Avenue, Boston, MA 02115（美国）][d]巴塞罗那大学巴塞罗那科技园，Baldiri Reixac, 10，科学技术服务博士M. Gair核磁共振设施，08028巴塞罗那（西班牙）本文的支持信息可在WWW上通过http://dx.doi.org/10.1002/cbic.200900575获得。
NMR spectroscopy is a useful tool for the study of protein structure, protein dynamics and molecular recognition processes, including both protein–protein and protein–ligand interactions. 4] However, the application of NMR experiments to large proteins remains a challenge. Transverse relaxation processes are accelerated as the size of the macromolecule grows and perdeuteration of proteins with low tumbling rates may be required. Thus, cells must be grown in D2O, [5] which, in general, reduces protein expression levels and significantly raises the cost of the NMR sample. Moreover, the assignment of spectra is limited by signal overlap, thus simplification of spectra by an appropriate selective labeling scheme is often required. 6] Selective labeling of specific amino acids can be achieved by using auxotrophic cell strains and adding the amino acid with a suitable isotope label to the medium. However, given that the biosynthetic pathways of amino acids are complex, cell growth may be limited when one of these pathways is disrupted, and this leads to lower expression levels. A less intrusive approach consists of the exploitation of the cell’s metabolic machinery to produce selectively labeled proteins and the particular choice of precursor determines whether a subset or a specific amino acid ends up labeled. Various authors have described such labeling approaches. As an example, if C-labeled a-ketobutyrate and a-ketoisovalerate are added to the growth medium, the cell then incorporates the C-label into valine, leucine and isoleucine sidechains. Similarly, addition of [2-C]or [4-C]-labeled indole to the medium allows the labeling of the tryptophan residues. Tryptophan, tyrosine and arginine are the most common amino acids in the hot spots of a given protein and are therefore involved in most of the binding energies in protein–protein and protein–ligand interactions. An inexpensive and reliable labeling strategy for the above-mentioned residues that is applicable to large proteins would be extremely useful. Here we report a cost-effective labeling strategy for the NMR study of large proteins. In this approach the N-label is selectively incorporated into the tryptophan side chains of the protein and the spectrum can be acquired without the need for deuteration. We applied this labeling to prolyl oligopeptidase (POP; EC 3.4.21.26), a serine protease of 80 kDa. In recent years, POP has gained relevance as a target for the treatment of cognitive disturbances. An array of strategies are currently being used to identify POP inhibitors, as these compounds show neuroprotective and cognition-enhancing effects in experimental animals. The X-ray structure of POP from porcine muscle revealed a distinctive two-domain structure: a catalytic domain with an a/b hydrolase fold and an unusual b-propeller domain. The costructure of POP in the presence of Z-prolyl-prolinal (ZPP), a canonical POP covalent inhibitor, shows that the specificity of the binding between the enzyme and the proline-containing inhibitor is provided by the hydrophobic interaction between POP tryptophan 595 (Trp595) and the ZPP proline ring. 16] The acquisition of a spectrum of a perdeuterated and uniformly N-labeled POP sample (U-[H,N]-POP; Figure 1 A) showed that even with perdeuteration and transverse relaxation optimized spectroscopy (TROSY), signal overlap was still a considerable handicap in cases with a large protein of 80 kDa. Therefore, a POP sample that was selectively labeled at tryptophan side chains (Trp[N-indole]-POP) was produced by supplementing the minimal growth medium with N-indole. The incorporation of the label was checked by mass spectrometry (Figure S1 in the Supporting Information). The resulting [H,N]-TROSY HSQC spectrum recorded with the Trp [NIndole]-POP sample showed a satisfactory signal-to-noise ratio (S/N) and eleven of the twelve expected tryptophan signals were present and well-dispersed (Figures 1 B, 2). It is noteworthy that despite of the high molecular weight of POP this result was obtained with a non-perdeuterated protein sample. The application of a previously described Trp[2-C-indole labeling was also evaluated. However, only few signals with poor S/N were detected in the [H,C]-aromatic TROSY spectrum acquired with a perdeuterated Trp[2-C-indole]-labeled POP sample (see SI). Thus, comparison of the results obtained for the selectively Trp[2-C-indole]and Trp[N-indole]-labeled POP shows that the Trp[N-indole] labeling is more convenient in cases with large proteins. This can be rationalized by considering the main differences between the TROSY effect of a H[a] Dr. T. Tarrag , Dr. B. Claasen, N. Kichik, Prof. E. Giralt Institute for Research in Biomedicine, Barcelona Science Park Baldiri Reixac, 10, 08028 Barcelona (Spain) Fax: (+ 34) 93-4037126 E-mail : ernest.giralt@irbbarcelona.org [b] Prof. E. Giralt Department of Organic Chemistry, University of Barcelona Mart Franqu s, 1, 08028 Barcelona (Spain) [c] Dr. R. A. Rodriguez-Mias Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115 (USA) [d] Dr. M. Gair NMR Facility, Scientific-Technical Services, University of Barcelona Barcelona Science Park, Baldiri Reixac, 10, 08028 Barcelona (Spain) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900575.
影响因子：
1.6
作者：
Studier, FW
通讯作者：
Studier, FW
使用氨基酸营养缺陷型菌株对重组蛋白进行选择性同位素标记。
DOI：
10.1007/978-1-59745-456-8_13
发表时间：
2007
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Whittaker,JamesW
通讯作者：
Whittaker,JamesW