Expanding the genetic code

Although chemists can synthesize virtually any small organic molecule, our ability to rationally manipulate the structures of proteins is quite limited, despite their involvement in virtually every life process. For most proteins, modifications are largely restricted to substitutions among the common 20 amino acids. Herein we describe recent advances that make it possible to add new building blocks to the genetic codes of both prokaryotic and eukaryotic organisms. Over 30 novel amino acids have been genetically encoded in response to unique triplet and quadruplet codons including fluorescent, photoreactive, and redox-active amino acids, glycosylated amino acids, and amino acids with keto, azido, acetylenic, and heavy-atom-containing side chains. By removing the limitations imposed by the existing 20 amino acid code, it should be possible to generate proteins and perhaps entire organisms with new or enhanced properties.     

Generation of a bacterium with a 21 amino acid genetic code

We have generated a completely autonomous bacterium with a 21 amino acid genetic code. This bacterium can biosynthesize a nonstandard amino acid from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon. The biosynthetic pathway for the amino acid p-aminophenylalanine (pAF) as well as a unique pAF synthetase and cognate tRNA were added to Escherichia coli. Denaturing gel electrophoresis and mass spectrometric analysis show that pAF is incorporated into myoglobin with fidelity and efficiency rivaling those of the common 20 amino acids. This and other such organisms may provide an opportunity to examine the evolutionary consequences of adding new amino acids to the genetic repertoire, as well as generate proteins with new or enhanced biological functions.     

Expanding the genetic code

The ability to incorporate unnatural amino acids into proteins directly in living cells will provide new tools to study protein and cellular function, and may generate proteins or even organisms with enhanced properties. Due to the limited promiscuity of some synthetases, natural amino acids can be substituted with close analogs at multiple sites using auxotrophic strains. Alternatively, this can be achieved by deactivating the editing function of some synthetases. The addition of new amino acids to the genetic code, however, requires additional components of the protein biosynthetic machinery including a novel tRNA-codon pair, an aminoacyl-tRNA synthetase, and an amino acid. This new set of components functions orthogonally to the counterparts of the common 20 amino acids, i.e., the orthogonal synthetase (and only this synthetase) aminoacylates the orthogonal tRNA (and only this tRNA) with the unnatural amino acid only, and the resulting acylated tRNA inserts the unnatural amino acid only in response to the unique codon. Using this strategy, the genetic code of Escherichia coli has been expanded to incorporate unnatural amino acids with a fidelity rivaling that of natural amino acids. This methodology is being applied to other cell types and unnatural analogs with a variety of functionalities.     

Biochemical analysis with the expanded genetic lexicon

The information used to build proteins is stored in the genetic material of every organism. In nature, ribosomes use 20 native amino acids to synthesize proteins in most circumstances. However, laboratory efforts to expand the genetic repertoire of living cells and organisms have successfully encoded more than 80 nonnative amino acids in E. coli, yeast, and other eukaryotic systems. The selectivity, fidelity, and site-specificity provided by the technology have enabled unprecedented flexibility in manipulating protein sequences and functions in cells. Various biophysical probes can be chemically conjugated or directly incorporated at specific residues in proteins, and corresponding analytical techniques can then be used to answer diverse biological questions. This review summarizes the methodology of genetic code expansion and its recent progress, and discusses the applications of commonly used analytical methods.     

Nonproteinogenic Amino Acid Building Blocks for Nonribosomal Peptide and Hybrid Polyketide Scaffolds

Freestanding nonproteinogenic amino acids have long been recognized for their antimetabolite properties and tendency to be uncovered to reactive functionalities by the catalytic action of target enzymes. By installing them regiospecifically into biogenic peptides and proteins, it may be possible to usher a new era at the interface between small molecule and large molecule medicinal chemistry. Site‐selective protein functionalization offers uniquely attractive strategies for posttranslational modification of proteins. Last, but not least, many of the amino acids not selected by nature for protein incorporation offer rich architectural possibilities in the context of ribosomally derived polypeptides. This Review summarizes the biosynthetic routes to and metabolic logic for the major classes of the noncanonical amino acid building blocks that end up in both nonribosomal peptide frameworks and in hybrid nonribosomal peptide‐polyketide scaffolds.     

Applications of hydrophilic interaction chromatography to amino acids,peptides, and proteins

This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed‐phase liquid chromatography, in a two‐dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited.     

Expanding the genetic code of an animal

Genetic code expansion, for the site-specific incorporation of unnatural amino acids into proteins, is currently limited to cultured cells and unicellular organisms. Here we expand the genetic code of a multicellular animal, the nematode Caenorhabditis elegans.     

A Synthetic Adenylation‐Domain‐Based tRNA‐Aminoacylation Catalyst

The incorporation of non‐proteinogenic amino acids represents a major challenge for the creation of functionalized proteins. The ribosomal pathway is limited to the 20–22 proteinogenic amino acids while nonribosomal peptide synthetases (NRPSs) are able to select from hundreds of different monomers. Introduced herein is a fusion‐protein‐based design for synthetic tRNA‐aminoacylation catalysts based on combining NRPS adenylation domains and a small eukaryotic tRNA‐binding domain (Arc1p‐C). Using rational design, guided by structural insights and molecular modeling, the adenylation domain PheA was fused with Arc1p‐C using flexible linkers and achieved tRNA‐aminoacylation with both proteinogenic and non‐proteinogenic amino acids. The resulting aminoacyl‐tRNAs were functionally validated and the catalysts showed broad substrate specificity towards the acceptor tRNA. Our strategy shows how functional tRNA‐aminoacylation catalysts can be created for bridging the ribosomal and nonribosomal worlds. This opens up new avenues for the aminoacylation of tRNAs with functional non‐proteinogenic amino acids.     

Wet and Functional Adhesives from One‐Step Aqueous Self‐Assembly of Natural Amino Acids and Polyoxometalates

Sessile organisms have undergone long‐term evolution to develop the unique ability by positioning themselves on wet solid surface through secreting adhesive proteins. The present study reveals that natural amino acid monomers can also exhibit similar adhesion capacity. This kind of biomimetic adhesives were created by the one‐step aqueous assembly of basic amino acids with assistance of anionic polyoxometalates. The polyoxometalates not only serve as multivalent scaffold to initiate the supramolecular cross‐linking of amino acid molecules, but also function as a redox component, bestowing the wet adhesives with electrochromic features.     

Characterization of N‐methylated amino acids by GC‐MS after ethyl chloroformate derivatization

Methylation is an essential metabolic process in the biological systems, and it is significant for several biological reactions in living organisms. Methylated compounds are known to be involved in most of the bodily functions, and some of them serve as biomarkers. Theoretically, all α‐amino acids can be methylated, and it is possible to encounter them in most animal/plant samples. But the analytical data, especially the mass spectral data, are available only for a few of the methylated amino acids. Thus, it is essential to generate mass spectral data and to develop mass spectrometry methods for the identification of all possible methylated amino acids for future metabolomic studies. In this study, all N‐methyl and N,N‐dimethyl amino acids were synthesized by the methylation of α‐amino acids and characterized by a GC‐MS method. The methylated amino acids were derivatized with ethyl chloroformate and analyzed by GC‐MS under EI and methane/CI conditions. The EI mass spectra of ethyl chloroformate derivatives of N‐methyl ( 1–18 ) and N,N‐dimethyl amino acids ( 19–35 ) showed abundant [M‐COOC₂H₅]⁺ ions. The fragment ions due to loss of C₂H₄, CO₂, (CO₂ + C₂H₄) from [M‐COOC₂H₅]⁺ were of structure indicative for 1–18 . The EI spectra of 19–35 showed less number of fragment ions when compared with those of 1–18 . The side chain group (R) caused specific fragment ions characteristic to its structure. The methane/CI spectra of the studied compounds showed [M + H]⁺ ions to substantiate their molecular weights. The detected EI fragment ions were characteristic of the structure that made easy identification of the studied compounds, including isomeric/isobaric compounds. Fragmentation patterns of the studied compounds ( 1–35 ) were confirmed by high‐resolution mass spectra data and further substantiated by the data obtained from ¹³C₂‐labeled glycines and N‐ethoxycarbonyl methoxy esters. The method was applied to human plasma samples for the identification of amino acids and methylated amino acids. Copyright © 2016 John Wiley & Sons, Ltd.     

Improving nature's enzyme active site with genetically encoded unnatural amino acids

The ability to site-specifically incorporate a diverse set of unnatural amino acids (>30) into proteins and quickly add new structures of interest has recently changed our approach to protein use and study. One important question yet unaddressed with unnatural amino acids (UAAs) is whether they can improve the activity of an enzyme beyond that available from the natural 20 amino acids. Herein, we report the >30-fold improvement of prodrug activator nitroreductase activity with an UAA over that of the native active site and a >2.3-fold improvement over the best possible natural amino acid. Because immense structural and electrostatic diversity at a single location can be sampled very quickly, UAAs can be implemented to improve enzyme active sites and tune a site to multiple substrates.     

Reevaluating Protein Photoluminescence: Remarkable Visible Luminescence upon Concentration and Insight into the Emission Mechanism

It is a textbook knowledge that protein photoluminescence stems from the three aromatic amino acid residues of tryptophan(Trp), tyrosine (Tyr), and phenylalanine (Phe), with predominant contributions from Trp. Recently, inspired by the intrinsic emission of nonaromatic amino acids and poly(amino acids) in concentrated solutions and solids, we revisited protein light emission using bovine serum albumin (BSA) as a model. BSA is virtually nonemissive in dilute solutions (≤0.1 mg mL^?1), but highly luminescent upon concentration or aggregation, showing unique concentration‐enhanced emission and aggregation‐induced emission (AIE) characteristics. Notably, apart from well‐documented UV luminescence, bright blue emission is clearly observed. Furthermore, persistent room‐temperature phosphorescence (p‐RTP) is achieved even in the amorphous solids under ambient conditions. This visible emission can be rationalized by the clustering‐triggered emission (CTE) mechanism. These findings not only provide an in‐depth understanding of the emissive properties of proteins, but also hold strong implications for further elucidating the basis of tissue autofluorescence.     

The graphical representation of protein sequences based on the physicochemical properties and its applications

Based on the chaos game representation, a 2D graphical representation of protein sequences was introduced in which the 20 amino acids are rearranged in a cyclic order according to their physicochemical properties. The Euclidean distances between the corresponding amino acids from the 2‐D graphical representations are computed to find matching (or conserved) fragments of amino acids between the two proteins. Again, the cumulative distance of the 2D‐graphical representations is defined to compare the similarity of protein. And, the examination of the similarity among sequences of the ND5 proteins of nine species shows the utility of our approach. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Adding L-3-(2-Naphthyl)alanine to the genetic code of E. coli

An unnatural amino acid, L-3-(2-naphthyl)alanine, has been site-specifically incorporated into proteins in Escherichia coli. An orthogonal aminoacyl-tRNA synthetase was evolved that uniquely aminoacylates the unnatural amino acid onto an orthogonal amber suppressor tRNA, which delivers the acylated amino acid in response to an amber nonsense codon with translational fidelity greater than 99%. This result, together with the in vivo site-specific incorporation of O-methyl-L-tyrosine reported previously, demonstrate that this methodology may be applicable to a host of amino acids. The expansion of the genetic code to include amino acids beyond the common 20 would provide an opportunity to better understand and possibly enhance protein (and perhaps organismal) function.     

Very efficient search for nucleotide alignments

We describe a very efficient search for nucleotide alignments, which is analogous to the novel very efficient search for protein alignment. Just as it has been the case with the alignment of proteins, based on 20 × 20 adjacency matrices for amino acids, obtained from a superposition of labeled amino acids adjacency matrices for the proteins considered, one can construct labeled matrices of size 4 × 4, listing adjacencies of nucleotides in DNA sequence. The matrix elements correspond to 16 pairs of adjacent nucleotides. To obtain DNA alignments, one combines information in the corresponding matrices for a pair of DNA nucleotides. Matrices are obtained by insertion of the sequential labels for pairs of nucleotides in the corresponding cells of the 4 × 4 tables. When two such matrices are superimposed, one can identify all segments in two DNA sequences, which are shifted relative to one another by the same amount in either direction, without using trial‐and‐error displacements of the two sequences one relative to the other to find local nucleotide alignments. © 2012 Wiley Periodicals, Inc.     

蛋白质功能化新策略:嵌入非天然氨基酸

天然蛋白质由20种天然氨基酸组成,这些蛋白质的构筑基元包含功能基团:羧基、氨基、巯基、硫醚、羟基、碱性胺、烷基和芳基。然而,这些有限的功能基团却不足以完成生物体内所有的生物学功能。为了更好地让生命的体现者--蛋白质完成更加精确和多样的生物学功能,自然界会对蛋白质进行翻译后的修饰,包括:磷酸化,甲基化,乙酰化或者羟基化,甚至在某些情况下,进化出一种新型的翻译机制以便插入硒代半胱氨酸或者吡咯霉素。受此启发,生物化学家发展出各种生物或化学方法来改变或插入新的蛋白质构筑基元,使天然蛋白质完成其相应的生物学功能或者使其具有某些特殊的性质,甚至是创造一种新酶。该文将简单介绍这些蛋白质修饰策略以及该领域的最新进展。     

Separation of amino acids by ion mobility spectrometry

The mobilities of the 20 common amino acids were determined by electrospray ionization ion mobility spectrometry. It was found that each amino acid had a different drift time and hence a different reduced mobility constant K0. This difference in drift time was less than 0.1 ms in many cases. With the instrument used in this study it would not be possible to resolve mixtures of some of the amino acids. It would however be possible to determine any single amino acid. In addition, the detection limits were determined for the 20 amino acids. They ranged from 50 to 700 pg. This indicates that the detection limits were less than 3 pmol for all of the amino acids and that many amino acids had detection limits less than 1 pmol.     

Homochirality and life

Before the emergence of life, left-handed amino acids (L-enantiomers) were selected and right-handed amino acids (D-enantiomers) were eliminated on the primal earth. Nevertheless, with the progress of analytical methods, D-amino acids have recently been found in higher order living organisms in the form of free amino acids, peptides, and proteins. Free D-amino acids have numerous physiological functions. D-amino acids containing animal peptides are well known as opioid peptides. D-amino acids in protein are related to aging. In this review, we describe the D-amino acids that are present and function as D-amino acid biosystems in our bodies.     

Minimal Tags for Rapid Dual‐Color Live‐Cell Labeling and Super‐Resolution Microscopy

The growing demands of advanced fluorescence and super‐resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue‐specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine‐functionalized organic dyes by the inverse‐electron‐demand Diels–Alder cycloaddition (SPIEDAC). Furthermore, we fine‐tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus‐like particles (VLPs) with dual‐color super‐resolution microscopy.