Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: minimizing misacylation

The incorporation of unnatural amino acids site-specifically is a valuable technique for structure-function studies, incorporation of biophysical probes, and determining protein-protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells.     

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 2: evaluating suppression efficiency

The incorporation of unnatural amino acids into proteins is a valuable tool for addition of biophysical probes, bio-orthogonal functionalities, and photoreactive cross-linking agents, although these approaches often require quantities of protein that are difficult to access with chemically aminoacylated tRNAs. THG73 is an amber suppressor tRNA that has been used extensively, incorporating over 100 residues in 20 proteins. In vitro studies have shown that the Escherichia coli Asn amber suppressor (ENAS) suppresses better than THG73. However, we report here that ENAS suppresses with <26% of the efficiency of THG73 in Xenopus oocytes. We then tested the newly developed Tetrahymena thermophila Gln amber suppressor (TQAS) tRNA library, which contains mutations in the second to fourth positions of the acceptor stem. The acceptor stem mutations have no adverse effect on suppression efficiency and, in fact, can increase the suppression efficiency. Combining mutations causes an averaging of suppression efficiency, and increased suppression efficiency does not correlate with increased DeltaG of the acceptor stem. We created a T. thermophila opal suppressor, TQOpS', which shows approximately 50% suppression efficiency relative to THG73. The TQAS tRNA library, composed of functional suppressor tRNAs, has been created and will allow for screening in eukaryotic cells, where rapid analysis of large libraries is not feasible.     

Position-specific incorporation of biotinylated non-natural amino acids into a protein in a cell-free translation system

Biotinylation is useful for the detection, purification and immobilization of proteins. It is performed by chemical modification, although position-specific and quantitative biotinylation is rarely achieved. We developed a position-specific biotinylation method using biotinylated non-natural amino acids. We showed that biotinylated p-aminophenylalanine derivatives were incorporated into a protein more efficiently than biotinylated lysine derivatives in a cell-free translation system. In addition, the biotinylated p-aminophenylalanines overcame the serious position-dependency observed for biotinylated lysines. The present method will be useful for detection and purification of proteins along with comprehensive exploration of surface-exposed residues and oriented immobilization of proteins.     

Multiple incorporation of non-natural amino acids into a single protein using tRNAs with non-standard structures

The ability to introduce non-natural amino acids into proteins opens up new vistas for the study of protein structure and function. This approach requires suppressor tRNAs that deliver the non-natural amino acid to a ribosome associated with an mRNA containing an expanded codon. The suppressor tRNAs must be absolutely protected from aminoacylation by any of the aminoacyl-tRNA synthetases in the protein synthesizing system, or a natural amino acid will be incorporated instead of the non-natural amino acid. Here, we found that some tRNAs with non-standard structures could work as efficient four-base suppressors fulfilling the above orthogonal conditions. Using these tRNAs, we successfully demonstrated incorporation of three different non-natural amino acids into a single protein.     

Site-specific incorporation of unnatural amino acids into urate oxidase in Escherichia coli

Urate oxidase catalyzes the oxidation of uric acid with poor solubility to produce 5-hydroxyisourate and allantoin. Since allantoin is excreted in vivo, urate oxidase has the potential to be a therapeutic target for the treatment of gout. However, its severe immunogenicity limits its clinical application. Furthermore, studies on the structure-function relationships of urate oxidase have proven difficult. We developed a method for genetically incorporating p-azido-L-phenylalanine into target protein in Escherichia coli in a site-specific manner utilizing a tyrosyl suppressor tRNA/aminoacyl-tRNA synthetase system. We substituted p-azido-L-phenylalanine for Phe(170) or Phe(281) in urate oxidase. The products were purified and their enzyme activities were analyzed. In addition, we optimized the system by adding a "Shine-Dalgarno (SD) sequence" and tandem suppressor tRNA. This method has the benefit of site-specifically modifying urate oxidase with homogeneous glycosyl and PEG derivates, which can provide new insights into structure-function relationships and improve pharmacological properties of urate oxidase.     

Regulation of protein synthesis by branched-chain amino acids in vivo

Recent advances in the understanding of mRNA translation have facilitated molecular studies on the regulation of protein synthesis by nutrients and the interplay between nutrients and hormonal signals. Numerous reports have established that, in skeletal muscle, the branched-chain amino acids (BCAAs) have the unique ability to initiate signal transduction pathways that modulate translation initiation. Of the BCAAs, leucine is the most potent. Oral administration of leucine to food-deprived rats enhances muscle protein synthesis, in part, through activation of the mRNA binding step of translation initiation. Interestingly, leucine signaling in skeletal muscle differs from that in liver, suggesting that the responses may be tissue specific. The purpose of this paper was to briefly review the current knowledge of how BCAAs act as regulators of protein synthesis in physiologically important tissues, with particular focus on the mechanisms by which BCAAs regulate translation initiation.     

Grouping of amino acids and recognition of protein structurally conserved regions by reduced alphabets of amino acids

Sequence alignment is a common method for finding protein structurally conserved/similar regions. However, sequence alignment is often not accurate if sequence identities between to-be-aligned sequences are less than 30%. This is because that for these sequences, different residues may play similar structural roles and they are incorrectly aligned during the sequence alignment using substitution matrix consisting of 20 types of residues. Based on the similarity of physicochemical features, residues can be clustered into a few groups. Using such simplified alphabets, the complexity of protein sequences is reduced and at the same time the key information encoded in the sequences remains. As a result, the accuracy of sequence alignment might be improved if the residues are properly clustered. Here, by using a database of aligned protein structures (DAPS), a new clustering method based on the substitution scores is proposed for the grouping of residues, and substitution matrices of residues at different levels of simplification are constructed. The validity of the reduced alphabets is confirmed by relative entropy analysis. The reduced alphabets are applied to recognition of protein structurally conserved/similar regions by sequence alignment. The results indicate that the accuracy or efficiency of sequence alignment can be improved with the optimal reduced alphabet with N around 9.     

Grouping of amino acids and recognition of protein structurally conserved regions by reduced alphabets of amino acids

Sequence alignment is a common method for finding protein structurally conserved/similar regions. However, sequence alignment is often not accurate if sequence identities between to-be-aligned sequences are less than 30%. This is because that for these sequences, different residues may play similar structural roles and they are incorrectly aligned during the sequence alignment using substitution matrix consisting of 20 types of residues. Based on the similarity of physicochemical features, residues can be clustered into a few groups. Using such simplified alphabets, the complexity of protein sequences is reduced and at the same time the key information encoded in the sequences remains. As a result, the accuracy of sequence alignment might be improved if the residues are properly clustered. Here, by using a database of aligned protein structures (DAPS), a new clustering method based on the substitution scores is proposed for the grouping of residues, and substitution matrices of residues at different levels of simplification are constructed. The validity of the reduced alphabets is confirmed by relative entropy analysis. The reduced alphabets are applied to recognition of protein structurally conserved/similar regions by sequence alignment. The results indicate that the accuracy or efficiency of sequence alignment can be improved with the optimal reduced alphabet with N around 9. Supported by the National Natural Science Foundation of China (Grant Nos. 90403120, 10474041 and 10021001) and the Nonlinear Project (973) of the NSM     

Concentrations of endogenous free amino acids in rabbit reticulocyte lysates and the effect of incubation

Amino acid analyses show that while the free amino acids found in the rabbit reticulocyte translation system do not increase during nuclease treatment or during prolonged storage, the endogenous levels of many amino acids are so high that the choice of a radioactive; precursor for a translation should be based not only on the abundance of the amino acid in the translation product but also on its concentration in the lysate preparation. It is shown that the variation of amino acid concentrations between different lysates is sufficiently small to allow one to use the concentrations reported in this study to calculate the amount of radioactive amino acid necessary for satisfactory incorporation.     

Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon

Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.     

Incorporation of glycosylated amino acid into protein by an in vitro translation system

O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.     

Beneficial role of amino acids in mitigating cytoskeletal actin glycation and improving F-actin content: In vitro

Aims: The actin filaments present in circulating leukocytes facilitate their passage through microvenules and capillaries by helping in their deformability. Decreased deformability of granulocytes is now known to cause occlusion of the retinal microcapillaries leading to hypoxia and the subsequent development of diabetic retinopathy. Structural and functional loss of proteins, due to non-enzymatic glycation and glycoxidation, has been reported to cause diabetic pathogenesis. As amino acids have been earlier reported to have antidiabetic properties, the present study involves the investigation of the susceptibility of the cytoskeletal actin to glycation and its mitigation by free amino acids. This study also involves quantifying F-actin in cultured mononuclear cells obtained from diabetic and normal healthy volunteers and on the effect of glucose and free amino acids on F-actin content. Methods: Commercial non-muscle actin and actin immuno-pre-cipitated from granulocytes obtained from (a) normal healthy human volunteers and (b) patients with type 2 diabetes mellitus were subjected to glycation studies using [U] ¹⁴C glucose. The effect of free amino acids, as antiglycating agents, was determined using various concentrations of lysine, arginine, alanine, aspartic acid and glutamic acid. F-actin content in cultured mononuclear cells was estimated by flow cytometry using fluorescein isothiocynate (FITC)-Phalloidin. Results: Commercial actin at physiological conditions of pH and temperature was found to undergo non-enzymatic glycation. The extent of in vitro glycation was significantly low (P<0.001) in actin isolated from patients with type2 diabetes when compared to the non-diabetic group, suggesting an increased in vitro structural modification of actin in patients with diabetes. All the free amino acids tested were found to have varying degrees of antiglycating effect. The F-actin content in the intact mononuclear cells obtained from diabetic patients was found to be low when compared with normal healthy volunteers (P<0.001). Similarly the F-actin content was significantly low when the normal mononuclear cells were incubated with glucose. This effect was reversed upon the addition of free amino acids to the incubation mixture. Conclusions: Free amino acids can play a positive role in improving leukocyte deformability by mitigating cytoskeletal actin glycation and improving F-actin content.     

Gas chromatographic determination of total amino acids and protein in sediments using the ninhydrin-CO2 reaction

A sensitive modification of the ninhydrin-CO₂ method involving the gas chromatographic determination of the total protein and amino acid content of sediment is described. The method gives a linear response over the amino acid concentration range 10^–5 M to 4 × 10^–2 M. It can be used for whole sediment, hydrolysates and interstitial water. The performance of the method is compared with the fluorescamine method for primary amines.     

Letter codes, structures, masses, and derivatives of amino acids

The structures and masses of amino acids and their more common modified forms (posttranslational and artificial) are presented as aids to analysis of polypeptides by mass spectrometry.     

Evaluation of the environmental implications of the incorporation of feed-use amino acids in the manufacturing of pig and broiler feeds using Life Cycle Assessment

The incorporation of feed-use (FU) amino acids (AAs) in diets results in a reduced use of protein-rich ingredients such as soybean meal, recognized to have elevated contributions to environmental impacts. This study investigated whether the incorporation of L-lysine.HCl, L-threonine and FU-methionine reduces the environmental impacts of pig and broiler feeds using Life Cycle Assessment. The following impact categories were considered: climate change, eutrophication, acidification, terrestrial ecotoxicity, cumulative energy demand and land occupation. Several feeds were formulated either to minimize the cost of the formulation (with or without AA utilization), to maximize AA incorporation (i.e. the cost of AA was considered to be similar to that of soybean meal), or to minimize greenhouse gas emissions. For both pig and broiler feeds, calculations were made first using only cereals and soybean meal as main ingredients and then using cereals and several protein-rich ingredients (soybean meal, rapeseed meal and peas). In addition, these calculations were performed using two types of soybean meal (from Brazil, associated with recent deforestation or not). For broiler feeds, two types of maize (from France, irrigated, with mineral fertilization v. not irrigated, with animal manure fertilization) were also tested. Regarding the feeds formulated to minimize cost, incorporation of AA decreased the values for eutrophication, terrestrial ecotoxicity and cumulative energy demand of both pig and broiler feeds, regardless of the base ingredients. Reduction in climate change and acidification due to the incorporation of AA depended on the nature of the feed ingredients, with the effect of AA incorporation being greater when combined with ingredients with high impacts such as soybean meal associated with deforestation. Feeds formulated to maximize AA incorporation generally had a similar composition to those formulated to minimize cost, suggesting that the costs of AA were not the limiting factor in their incorporation. Feeds formulated to minimize greenhouse gas emissions had the lowest values for climate change and cumulative energy demand, but not for other impacts. Further research is needed to elucidate whether the incorporation of additional AA (tryptophan and valine) along with L-lysine, L-threonine and FU-methionine could decrease on the environmental impacts of pig and broiler feeds further.     

An analysis of intron positions in relation to nucleotides, amino acids, and protein secondary structure

We present an analysis of intron positions in relation to nucleotides, amino acid residues, and protein secondary structure. Previous work has shown that intron sites in proteins are not randomly distributed with respect to secondary structures. Here we show that this preference can be almost totally explained by the nucleotide bias of splice site machinery, and may well not relate to protein stability or conformation at all. Each intron phase is preferentially associated with its own set of residues: phase 0 introns with lysine, glutamine, and glutamic acid before the intron, and valine after; phase 1 introns with glycine, alanine, valine, aspartic acid, and glutamic acid; and phase 2 introns with arginine, serine, lysine, and tryptophan. These preferences can be explained principally on the basis of nucleotide bias at intron locations, which is in accordance with previous literature. Although this work does not prove that introns are inserted into genomes at specific proto-splice sites, it shows that the nucleotide bias surrounding introns, however it originally occurred, explains the observed correlations between introns and protein secondary structure.     

In vitro prediction of standardised ileal crude protein and amino acid digestibilities in grain legumes for growing pigs

The study was conducted to validate in vitro prediction of standardised ileal digestibilities (SID) of crude protein (CP) and amino acids (AA) in grain legumes for growing pigs using six different cultivars of faba beans (Vicia faba), six different cultivars of field peas (Pisum sativum), and five different cultivars of lupins (Lupinus spp.). The SID for CP and AA were predicted from in vitro analysis by means of a two-step enzymatic method using pepsin and pancreatin incubations. In vitro predicted SID values of CP and AA were generally higher than the corresponding SID values measured in vivo. There were strong linear relationships (r2 = 0.73 for Lys to r2 = 0.91 for Cys and Trp) between in vivo and in vitro predicted SID values in the assay feed ingredients if grain legume species (i.e. faba beans, field peas and lupins) was included as a covariate in multiple linear regression analysis. However, to rapidly and accurately predict SID of CP and AA in individual batches of various feed ingredients, further studies are warranted.     

In Vitro Protein Import of a Putative Amino Acid Transporter from Arabidopsis thaliana into Chloroplasts and Its Suborganellar Localization

We identified a gene product of At5g19500 (At5g19500p) from Arabidopsis thaliana that is homologous to EcTyrP, a tyrosine-specific transporter from Escherichia coli. Computational analyses of the amino acid sequence of At5g19500p predicted 11 transmembrane domains (TMDs) and a potential plastid targeting signal at its amino terminus. As a first step toward understanding the possible role of At5g19500p in plant cells, we attempted to determine the localization of At5g19500p by an in vitro chloroplastic import assay using At5g19500p translated in a cell-free wheat germ system (Madin et al., Proc. Natl. Acad. Sci. USA, 97, 559–564 (2000)), followed by subfractionation of the chloroplasts. At5g19500p was successfully imported into chloroplasts, and the newly transported mature form of At5g19500p was recovered from the inner envelope membrane.