Site-specific incorporation of PEGylated amino acids into proteins using nonnatural amino acid mutagenesis

Site-directed incorporation of PEGylated nonnatural amino acids with 4, 8, and 12 repeated ethylene glycol units was examined in a cell-free translation system. PEGylated aminophenylalanine derivatives were successfully incorporated into proteins, whereas PEGylated lysines were not. The incorporation efficiency of the PEGylated amino acids decreased with an increase in PEG chain length. The present method will be useful for preparation of proteins which are PEGylated in a site-specific and quantitative manner.     

Incorporation of nonnatural amino acids into proteins by using five-base codon-anticodon pairs.

A novel strategy for the incorporation of nonnatural amino acids into proteins was developed by using five-base codon-anticodon pairs. The streptavidin mRNA containing five-base codon CGGUA and the chemically aminoacylated tRNA with five-base anticodon UACCG were prepared, and added into E. coli in vitro translation system. As a result, the nonnatural amino acid was successfully incorporated into desired position of the protein. Other five-base codons CGGN1N2, where N1 and N2 indicate one of four nucleotides, were also available for the incorporation of the nonnatural amino acid.     

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.     

Five-base codons for incorporation of nonnatural amino acids into proteins

Extension of the genetic code for the introduction of nonnatural amino acids into proteins was examined by using five-base codon–anticodon pairs. A streptavidin mRNA containing a CGGUA codon at the Tyr54 position and a tRNA_UACCG chemically aminoacylated with a nonnatural amino acid were added to an Escherichia coli in vitro translation system. Western blot analysis indicated that the CGGUA codon is decoded by the aminoacyl-tRNA containing the UACCG anticodon. HPLC analysis of the tryptic fragment of the translation product revealed that the nonnatural amino acid was incorporated corresponding to the CGGUA codon without affecting the reading frame adjacent to the CGGUA codon. Another 15 five-base codons CGGN₁N₂, where N₁ and N₂ indicate one of four nucleotides, were also successfully decoded by aminoacyl-tRNAs containing the complementary five-base anticodons. These results provide a novel strategy for nonnatural mutagenesis as well as a novel insight into the mechanism of frameshift suppression.     

Fluorescent labeling of cell-free synthesized proteins by incorporation of fluorophore-conjugated nonnatural amino acids

Although fluorescent dyes, such as fluorescein derivatives, have bulky and complex structures, nonnatural amino acids carrying these fluorescein derivatives are acceptable by the Escherichia coli ribosome and are useful for the cotranslational fluorescent labeling of cell-free synthesized proteins. Surprisingly, the incorporation efficiency of nonnatural amino acids carrying fluorescein derivatives into translated proteins depends on the source of the translational machinery used in cell-free protein synthesis. That is, whereas the E. coli ribosome efficiently supported the incorporation of nonnatural amino acids carrying fluorescein derivatives into a protein structure, no detectable fluorescent signal was observed from the protein expressed in the eukaryotic cell-free protein synthesis system performed in the presence of fluorescein-conjugated aminoacylated transfer RNA (tRNA).     

Incorporation of two nonnatural amino acids into proteins through extension of the genetic code.

A novel method of the in vitro incorporation of two nonnatural amino acids into proteins through extension of the genetic code was developed. The streptavidin mRNA containing AGGU and CGGG, and chemically aminoacylated tRNA(ACCU) and tRNA(CCCG) were prepared, then they were added into E. coli in vitro protein synthesizing system. As a result, two nonnatural amino acids were successfully incorporated into desired sites of streptavidin.     

Conformational studies of nucleic acids: III. Empirical multiple correlation functions for nucleic acid torsion angles

There are seven significantly variable torsion angles in each monomer unit of a polynucleotide. Because of this, it is computationally infeasible to consider the energetics of all conformations available to a nucleic acid without the use of simplifications. In this paper, we develop functions suggested by and regression fit to crystallographic data which allow three of these torsion angles, alpha (O3'-P-O5'-C5'), delta (C5'-C4'-C3'-O3') and epsilon (C4'-C3'-O3'-P), to be calculated as dependent variables of those remaining. Using these functions, the seven independent torsions are reduced to four, a reduction in complexity sufficient to allow an examination of the global conformational energetics of a nucleic acid for the remaining independent torsion angles. These functions are the first to quantitatively relate a dependent nucleic acid torsion angle to several different independent angles. In all three cases the data are fit reasonably well, and in one case, alpha, the fit is exceptionally good, lending support for the suitability of the functions in conformational searches. In addition, an examination of the most significant terms in each of the correlation functions allows insight into the physical basis for the correlations.     

Random insertion and deletion mutagenesis for construction of protein library containing nonnatural amino acids.

A new method was developed for the generation of a library of mutant proteins that contained nonnatural amino acids. The method, "random insertion and deletion (RID) mutagenesis", is based on the deletion of an arbitrary number of bases at random positions and, at the same time, the insertion of an arbitrary sequence into the same position. By using this method, randomly selected three consecutive bases in the gene of green fluorescence protein (GFP) were replaced by a CGGT 4-base codon. When this DNA library was expressed in E. coli, about 80% of colonies lost the fluorescence. The non-fluorescent colonies were picked up and the genes were sequenced. Replacement of three consecutive bases by CGGT 4-base codon was found in two of the four mutated genes.     

Dynamic properties of interaction between nucleic acid bases and models of amino acids

We have discussed the possible hydrogen bonding mode of adenine-uracil base dimers and peptide side chains, together with their characteristic effects on the stability of adenine-uracil base pairing in chloroform solution by using proton nuclear magnetic resonance methods, where acetamide and butyric acid were used as a model of residues of glutamine and glutamic acid, respectively, and were added to an adenine-uracil equimolar mixture. The stability of base pairing was affected significantly when the model compounds approached the adenine-uracil base pairs; that is, the Hoogsteen type was destroyed, while the Watson-Crick type was formed more favorably.     

Effect of season and artificial diet on amino acids and nucleic acids in gonads of green sea urchin Strongylocentrotus droebachiensis

The content of total and free amino acids (FAA) in green sea urchin (Strongylocentrotus droebachiensis) gonads varied with the season and feeding on an artificial diet. Glycine was the dominant amino acid in each season contributing 12.9-16.6% to the total amino acid (TAA) content, peaking in the spring. In the FAA profile, glycine accounted for 30.3-61.4% in different seasons. A grain-based artificial diet had noticeable effects on the total and FAA compositions of S. droebachiensis. Although, glycine was the dominant amino acid in the TAA profile during early harvesting, tyrosine in gonads became more dominant on week 9 of feeding. Furthermore, glycine was the dominant amino acid in the FAA pool after feeding the artificial diet. The total FAA content in the gonads increased significantly (P<0.05) from 20.6 on week 0 to 180.6 mg/g dry mass tissue on week 3. There were no significant (P<0.05) changes between week 6 and week 9. Deoxyribonucleic acid (DNA) content exceeded that of ribonucleic acid (RNA) in each season, while in cultured urchins, RNA content exceeded that of DNA only on week 6. The RNA/DNA ratio was significantly increased in the summer, whereas this ratio was increased up to week 6 followed by a decrease on week 9 in cultured counterparts.     

Contribution of hydrolyzed nucleic acids and their constituents to the apparent amino acid composition of biological compounds.

Acid hydrolysis of protein-free mixtures of nucleotides, nucleosides, and nucleic acids yields amino acids, free bases, and possibly other unidentified fragments when analyzed by thin-layer chromatography and by standard amino acid analysis. Glycine is the predominant amino acid detected, which may constitute 47–97% of the apparent amino acid composition, depending on the type of material subjected to hydrolysis. Obviously, hydrolyzed nucleic acids or their constituents can therefore contribute to the apparent amino acid composition of a supposedly pure peptide or of other more complex mixtures of compounds mistakenly believed to contain only protein. To circumvent this problem, we suggest that nucleotides or nucleic acid moieties should be removed from any product for which the amino acid composition is desired, and that whenever a large glycine peak is noted in a hydrolyzed sample, the presence of nucleic acids or their constituents should be suspected.     

Theoretical studies on protein-nucleic acid interactions. I. Interaction of positively charged amino acids with nucleic acid fragments

Coulombic interactions between the side chains of charged amino acids (Arg⁺, Lys⁺, and His⁺) and negatively charged phosphate groups of nucleic acid fragments have been studied theoretically. Diribose monophosphate and dideoxyribose monophosphate are chosen as model systems for single-stranded RNA and DNA, respectively. The interaction energies have been calculated by second-order perturbation theory using simplified formulas for individual terms. The interaction energy in this formalism is a sum of electrostatic, polarization, dispersion, and repulsive energies. Our results show that about 90% of the total interaction energy is contributed by the electrostatic term alone. Contribution from the repulsive term exceeds that from the dispersion term. Calculated interaction energies suggest that Lys⁺ and His⁺ form more stable complexes with RNA than with single-stranded DNA. On the other hand, Arg⁺ has a higher affinity for DNA than for RNA. The affinity of nucleic acids for the three amino acids is in the order Lys⁺ > His⁺ > Arg⁺. Further, the basic amino acid residues form more stable complexes with A-DNA than with B-DNA. The role of the Coulombic interactions in the specific recognition of nucleic acids by proteins is discussed.     

Free amino acids and nucleic acid content of cell nuclei isolated by a modification of Behrens' technique

1. Nuclei were prepared from frozen rat liver by a modification of the technique of Behrens, and were studied with regard to the content of free amino acids and nucleic acid. 2. Under rigorously controlled conditions, preparations of nuclei are obtained by the Behrens' method which form a gel in the presence of 5 or 10 per cent NaCl or of water plus a small amount of dilute alkali; whereas when conditions are less rigorously controlled, nuclei are obtained which form no such gel. The property of forming gels with alkali is probably characteristic of all cell nuclei which have not undergone autolysis. 3. Nuclei prepared by the Behrens' technique contain the enzymes arginase, catalase, and esterase in very appreciable concentrations. 4. The free amino acids of the isolated cell nuclei, as well as of other liver cell fractions, have been investigated using the technique of paper chromatography. 5. The chromatographic patterns of the free amino acids of whole cells, ground cytoplasm, and isolated cell nuclei were very similar or identical. A feature of interest in these chromatograms was the faintness or absence of the spots due to a number of the essential amino acids, as compared to the intensities of the spots due to glycine, alanine, and glutamic acid. Glutathione was present in the isolated nuclei as well as in the whole cells. 6. Chromatograms made from hydrolysates of nuclei showed high concentrations of the essential amino acids and were similar to chromatograms of hydrolysates of typical proteins.     

Involvement of basic amino acids in the activity of a nucleic acid helix-destabilizing protein

Under conditions of low ionic strength, ribonuclease A, which binds more tightly to single- than to double-stranded DNA, lowers the melting temperature of DNA helices (Jensen and von Hippel (1976) J. Biol. Chem. 251, 7198-7214). The effects of chemical modification of lysine and arginine residues on the helix-destabilizing properties of this protein have been examined. Removal of the positive charge on the lysine epsilon-amino group, either by maleylation or acetylation, destroys the ability of RNAase A to lower the Tm of poly[d(A-T)]. However, reductive alkylation of these residues, which has not effect on charge, yields derivatives which lower the Tm by only about one-half that seen with unmodified controls. Phenylglyoxalation of arginines can largely remove the Tm-depressing activity of RNAase A. RNAase S, which is produced by cleavage of RNAase A between amino acids 20 and 21, possesses DNA helix-destabilizing activity comparable to that of the parent protein, whereas S-protein (residues 21-124) increases poly[d(A-T)] Tm and S-peptide (1-20) has no effect on Tm. These results suggest that specific location of several basic amino acids situated on the surface of RNAase A is largely responsible for this protein's DNA melting activity.