In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy
In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy
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DOI：
10.1021/ja801602q
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发表时间：
2008-07-23
影响因子：
15
通讯作者：
Geierstanger, Bernhard H.
中科院分区：
文献类型：
--
作者：
Cellitti, Susan E.;Jones, David H.;Geierstanger, Bernhard H.
在体内掺入同位素标记的非天然氨基酸到大蛋白质大大降低了核磁共振（NMR）谱的复杂性。通过共表达对添加到培养基中的非天然氨基酸具有特异性的正交tRNA/氨酰-tRNA合成酶对和在所需掺入位点具有TAG琥珀密码子的目的蛋白质来实现掺入。为了证明该方法用于NMR研究的实用性，将2-氨基-3-（4-（三氟甲氧基）苯基）丙酸（OCF（3）Phe）、C-13/N-15标记的对甲氧基苯丙氨酸（OMePhe）和N-15标记的邻硝基苄基酪氨酸（oNBTyr）分别掺入人脂肪酸合酶（FAS-TE）的33 kDa硫酯酶结构域的活性位点周围的11个位置。在此过程中，OCF 3 Phe进化出一种新的tRNA合成酶。通过在每个掺入质粒上包括相应氨酰-tRNA合成酶基因的诱导型拷贝来提高掺入效率和FAS-TE产率。仅使用8至25 mg的非天然氨基酸，通常从在丰富培养基中生长的50 mL大肠杆菌培养物中产生足以用于一个0.1 mM NMR样品的2 mg FAS-TE。然后使用单标记的蛋白质样品来研究工具化合物的结合。不同单位点突变体的H-1-N-15 HSQC、H-1-C-13 HSQC和F-19 NMR光谱中的化学位移变化一致地鉴定了结合位点和配体结合对一些残基的构象交换的影响。（3）活性位点酪氨酸的Phe突变体抑制结合;通过UV切割来自oNBTyr的硝基苄基-光笼，在该位点掺入15个N-Tyr，重新建立结合。这些数据不仅表明了使用非天然氨基酸通过NMR研究大蛋白质的稳健方法，而且还建立了一种新的途径，用于在不改变蛋白质序列的情况下在单个残基处对蛋白质进行位点特异性标记，这是目前无法用任何其他方法实现的壮举。
In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF(3)Phe), C-13/N-15-labeled p-methoxyphenylalanine (OMePhe), and N-15-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in H-1-N-15 HSQC, H-1-C-13 HSQC, and F-19 NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF(3)Phe mutants of an active site tyrosine inhibited binding; incorporating 15 N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.