Báo cáo khoa học: Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins

Translational Incorporation of L-3,4-dihydroxyphenylalanine into Proteins

Tác giả: Kiyoshi Ozawa, Madeleine J. Headlam, Dmitri Mouradov, Stephen J. Watt, Jennifer L. Beck, Kenneth J. Rodgers, Roger T. Dean, Thomas Huber, Gottfried Otting, Nicholas E. Dixon

Lĩnh vực: Sinh hóa, Sinh học phân tử

Nội dung tài liệu: Nghiên cứu này khảo sát việc đưa L-3,4-dihydroxyphenylalanine (DOPA) vào protein thông qua hệ thống tổng hợp protein không tế bào của vi khuẩn Escherichia coli, thay thế tyrosine bằng DOPA trong hỗn hợp phản ứng. Kết quả cho thấy DOPA được tích hợp đặc hiệu vào vị trí của tyrosine, đạt hiệu suất hơn 90% tại mỗi vị trí tyrosine. Điều này cho phép ghi nhận phổ NMR ¹⁵N-HSQC rõ ràng. Phương pháp nhuộm đặc hiệu DOPA đã được chứng minh là nhạy và ứng dụng rộng rãi để đánh giá sản xuất protein không tế bào. Trong số bốn protein được sản xuất dưới dạng hòa tan khi có mặt tyrosine, hai protein đã tạo thành các khối tập hợp không hòa tan khi có hàm lượng DOPA cao. DOPA được tìm thấy trong protein người, thường liên quan đến các trạng thái bệnh lý liên quan đến sự kết tụ protein và/hoặc gấp sai protein. Các phát hiện này gợi ý rằng protein gấp sai và kết tụ có thể là kết quả của việc tích hợp DOPA nội bào do stress oxy hóa, thông qua cơ chế gấp sai protein trên ribosome. Các hệ thống biểu hiện protein không tế bào năng suất cao rất phù hợp để thu thập thông tin nhanh chóng về độ hòa tan và sự kết tụ của các chuỗi polypeptide mới tổng hợp.

Mục lục chi tiết:

• Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins
• Keywords
• Correspondence
• (Received 16 February 2005, revised 2 April 2005, accepted 25 April 2005)
• doi:10.1111/j.1742-4658.2005.04735.x
• An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean ¹⁵N-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.
• The incorporation of non-natural amino acids opens up the possibility to endow proteins with properties that cannot be attained with the 20 natural amino acids encoded by DNA base triplets. The incorporation of non-natural amino acids can readily be achieved with the natural protein-translational machinery, if the structure of the modified amino acid is closely related to the natural amino acid, so that it can be loaded onto tRNA by one of the natural aminoacyl-tRNA synthetases. A wide range of non-natural amino acids has been incorporated into proteins in this way [1]. In general, the efficiency of incorporation decreases with increasing K_M value of the aminoacyl-tRNA synthetase for the respective amino acid. This holds, in particular, for the in vivo incorporation of non-natural amino acids, where a pool of natural amino acids is always present. This problem can be circumvented by the use of auxotrophic strains [1] or cell-free protein production systems derived from nonauxotrophic strains combined with a suitably manipulated medium for protein synthesis [2,3]. Recently, high-yield, cell-free protein production systems have become available that allow the synthesis of proteins in quantities sufficient for structural genomics applications [4–7]. High-level incorporation of seleno-methionine (Se-Met) for X-ray crystallography and fluoro-tryptophan (F-Trp) for NMR has been demonstrated [8,9], but limited dilution of isotope-labelled with unlabelled natural amino acids has also been reported [6].
• This study investigated the high-level, high-yield incorporation of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixture of an Escherichia coli cell-free transcription/translation system. The K_M value for E. coli tyrosyl-tRNA synthetase (TyrRS) was reported to be ≈200-fold higher for L-DOPA than for L-tyrosine (1.4 mM vs. 6 μM) [10,11], i.e. the natural enzyme discriminates against DOPA one order of magnitude more strongly than the respective aminoacyl tRNA synthetases incorporating Se-Met vs. Met [12] and F-Trp vs. Trp [13]. DOPA-enrichment is advantageous as it allows the facile assessment of protein production levels, because a highly specific staining method is available [14]. Finally, DOPA is produced naturally in humans by tyrosinase-catalysed oxidation of tyrosine in melanocytes for melanin production, and by tyrosine hydroxylase in the brain for biosynthesis of catecholamine neurotransmitters [15].
• In addition, the accumulation of protein-bound DOPA in cells and tissues is a feature of a number of pathologies associated with ageing, such as atherosclerosis [15] and cataractogenesis [16], where it derives at least in part from oxygen-radical mediated post-translational oxidation of tyrosine side chains in proteins [15]. It has recently been shown that DOPA can be incorporated directly from the medium into proteins in cultured mouse [16] and human [17] cells, and that incorporation relies on protein synthesis [16]. If translational (ribosome-mediated) incorporation of DOPA is a distinct possibility, the structural and functional consequences of DOPA incorporation would be important to assess.
• This study used a preparative E. coli cell-free transcription/translation system [5,18] to incorporate DOPA into four different in vitro-synthesized proteins. MS and NMR spectroscopy were used to verify whether DOPA incorporation occurred at positions normally occupied by tyrosine, and to assess the level of DOPA incorporation. The effects on folding of the four proteins (all of which have known structures) were assessed by examination of their solubility following their in vitro synthesis.
• Results
• Protein synthesis in the presence of DOPA
• The effect of substitution of DOPA for tyrosine was investigated in a preparative in vitro protein synthesis system that employs an E. coli cell-free (S30) extract as the source of ribosomes, aminoacyl-tRNA synthetases and translation factors [5,18]. We chose to examine the synthesis of four different proteins whose three-dimensional structures are known from X-ray crystallographic studies: the peptidyl-prolyl cis-trans isomerases E. coli (PpiB; Protein Data Bank Accession no. 2NUL) [19] and human cyclophilin A (hCypA; Protein Data Bank Accession no. 2CPL) [20], the E. coli flavohaemoglobin (HMP; Protein Data Bank Accession no. 1GVH) [21] and the Aequorea victoria green fluorescent protein (GFP; Protein Data Bank Accession no. 1EMA) [22]. The first two proteins had previously been shown to be produced in good yield in the in vitro reaction [5,6,18,23].
• All four proteins were found to be synthesized to similarly high levels in the presence of 1 mM tyrosine or DOPA (Fig. 1A; data not shown for PpiB). Analysis of the supernatant and pellet fractions by Coomassie Brilliant Blue staining of a SDS/PAGE gel indicated that all were soluble or mostly soluble when expressed using tyrosine (Fig. 1A). Whereas PpiB (18 kDa) and HMP (44 kDa) were still largely soluble when they were synthesized with DOPA, > 50% of hCypA (18 kDa) and GFP (27 kDa) were in the insoluble fraction. This implies that incorporation of DOPA can interfere with correct protein folding.
• The yield of PpiB depended remarkably little on the concentration of DOPA or tyrosine in the reaction mixtures. High yields similar to those obtained with 1 mM tyrosine or DOPA were obtained with either amino acid at 50 μM (data not shown). With 10 and 5 μM DOPA, the yields were ≈20 and 50% lower, respectively, than with 10 μM tyrosine, and there was still discernible production of PpiB when both amino acids were omitted. This is presumably because of the presence of a trace of tyrosine as a contaminant in the S30 extract or its biosynthesis during the reaction.
• DOPA is incorporated into proteins during cell-free synthesis
• To show that DOPA was incorporated into the translated proteins, we first used a redox staining method employing nitroblue tetrazolium (NBT), which detects proteins containing o-catechols, like DOPA, after their separation by SDS/PAGE and western transfer to poly(vinylidene difluoride) membranes [14,17]. The staining method was verified using purified His₆-PpiB that had been produced by cell-free synthesis in the presence of 0.05 or 1.0 mM DOPA or 1.0 mM tyrosine. The protein was purified in similar yields from each reaction (≈2 mg per 2 mL of reaction mixture) by metal-ion affinity chromatography and analysed by duplicate SDS/PAGE gels that were stained either with Coomassie Brilliant Blue (Fig. 2A) or by redox staining (Fig. 2B). Only His₆-PpiB that had been produced in the presence of DOPA stained with NBT, and the staining intensity was somewhat higher for the sample produced with 1.0 mM in comparison with 0.05 mM DOPA.
• This staining method could also be used to detect de novo synthesized proteins in the crude reaction mixtures. Proteins synthesized with tyrosine were not stained by NBT, but those made in the presence of DOPA were readily and specifically detected (Fig. 1B). These results show the incorporation of DOPA during cell-free protein synthesis. The sensitivity of this method is comparable with staining by Coomassie Brilliant Blue, and only newly synthesized proteins were detected, including some minor species presumed to have been produced by proteolysis and/or premature termination of translation (Fig. 1B). It confirmed that high-level DOPA incorporation results in mostly insoluble protein in the cases of GFP and hCypA and mostly soluble protein for HMP and PpiB. Given that the chromophore in GFP involves a tyrosine (Tyr66), and that its photophysical properties are particularly sensitive to substitution of this residue [22,24,25], it was of interest to examine fluorescence spectra of in vitro synthesized GFP. The excitation and emission spectra of crude mixtures containing the fraction of soluble GFP that had been produced with tyrosine or DOPA were found to be identical. Nevertheless, the yield of fluorescence was low (10–20%) in the soluble fraction from the DOPA sample compared with that prepared with tyrosine (data not shown). This indicates that although some portion of GFP was capable of folding correctly into a soluble form when DOPA was incorporated in place of Tyr66, the chromophore either did not form or was not appreciably fluorescent. The insoluble fraction was not noticeably fluorescent.
• Having shown that DOPA could be translationally incorporated into various proteins, we next established that this occurred specifically in place of tyrosine. This was done in three ways: (a) by showing that the mass of intact purified DOPA-His₆-PpiB, as determined by ESI-MS under native conditions, was increased by 16 mass units per tyrosine residue; (b) by showing that a relative increase in mass of tryptic fragments from DOPA-His₆-PpiB, as determined by ESI-MS after separation by RP-HPLC was observed only for peptides that would otherwise contain tyrosine and (c) that NMR chemical shift changes for samples of selectively ¹⁵N-labelled DOPA-PpiB relative to native ¹⁵N-labelled PpiB were consistent with the specific incorporation of DOPA in place of tyrosine.
• ESI-MS of DOPA-labelled His₆-PpiB
• The mass of purified His₆-PpiB produced in the presence of 0.05 mM DOPA was compared with that of the normal protein produced with 1.0 mM tyrosine. Two species were present in the tyrosine sample in almost equal proportions, with M_r values of 19 221.7 and 19 249.7 (Fig. 3A, peaks A and B, respectively). The larger component corresponds to a form of the protein that retains the N-formyl group on the N-terminal methionine residue (calculated M_r 19 249.6), and the smaller is the mature protein produced after deformylation (calculated M_r 19 221.6). This is in accord with the results of a previous NMR study, in which amide resonances could be observed for the N-terminal methionine as well as for the following residue in hCypA [18], indicating that our S30 extract is deficient in peptide deformylase activity [6].
• His₆-PpiB contains three tyrosine residues. When produced with DOPA, the protein contained several species (Fig. 3B). The most abundant had masses of 19 297.9 (peak G), 19 281.9 (peak F) and 19 269.3 (peak E), in order of decreasing intensity. These species correspond to the N-formylated protein with three and two, and deformylated protein with three DOPA residues, respectively. Semiquantitative assessment of the incorporation level of DOPA was made by comparison of the sum of the peak heights of the 3-DOPA species (peaks E, G, H and I) vs. the sum of the peak heights of the 2-DOPA species (peaks D and F). This ratio was found to be 3 : 1. The same ratio was found by comparing the peak heights of peaks E (2-DOPA species) and D (1-DOPA species). This suggests that about three-quarters of all tyrosine residues had been replaced by DOPA, corresponding to an incorporation level of > 90% at each of the three tyrosine sites. The less than 100% efficiency was presumably due to traces of tyrosine (and/or tyrosyl-tRNA) remaining in, or synthesized by, the cell-free extract.
• HPLC-ESI-MS of tryptic peptides from DOPA-labelled His₆-PpiB
• Peptides resulting from partial tryptic digestion of His₆-PpiB that had been produced using 1 mM tyrosine or 1 mM DOPA were separated by HPLC and analysed by in-line ESI-MS. Peptides of M_r > 500 (the threshold for ESI-MS), identified by correspondence between their M_r values and amino acid composition, spanned 165 (96%) of 172 residues of the amino acid sequence (Table 1). For both samples, a significant portion of the N-terminal peptides had a mass 28.0 units higher than expected; this confirms that the additional mass seen for much of the in vitro synthesized intact His₆-PpiB (Fig. 3B) is due to retention of the formyl group on the N-terminal methionine residue (see above) [6,18].
• Three tryptic peptides, each containing one of the three tyrosine residues in His₆-PpiB, were observed at the expected mass in both samples. In the sample prepared with DOPA, three additional, more-abundant, peptides were observed, each with masses 16.0 units higher than these three (Table 1), as might be expected if each of the tyrosine residues was substantially replaced by DOPA. No peptides were observed that might correspond to significant replacement of another amino acid residue by DOPA.
• NMR analysis of DOPA-labelled (¹⁵N)PpiB
• In a previous study [18], we showed that crude reaction mixtures containing hCypA produced in vitro in the presence of ¹⁵N-labelled amino acids could be used directly, after dialysis into an NMR buffer (phosphate, pH 6.5), to record residue-specific ¹⁵N-HSQC NMR spectra. The protein remained soluble during and following dialysis [18]. In contrast, in this study, the initially soluble portion of DOPA-hCypA precipitated quantitatively on dialysis into the NMR buffer, which indicates that incorporation of DOPA destabilizes the native structure of hCypA.
• Therefore, the E. coli homologue of hCypA, PpiB, was used for NMR studies. We recorded ¹⁵N-HSQC NMR spectra of crude mixtures containing PpiB that had been synthesized in the presence of tyrosine or DOPA using amino acid mixtures that contained both (¹⁵N)cysteine and (¹⁵N)phenylalanine in place of the corresponding unlabelled amino acids, and then dialysed into an NMR buffer (Fig. 4A). The buffer was identical to that used previously during assignment of the amide ¹