Site-Specific Introduction of Bioorthogonal Handles to Nanopores by Genetic Code Expansion

Site-specific functionalization of natural amino acid-containing biological nanopores is pivotal in single molecule sensing. However, pore engineering methodologies are restricted to a limited choice and introduction of unnatural chemical components is extremely difficult. Herein we report the genetic code expansion (GCE) strategy to introduce unnatural amino acid (UAA) to an octameric Mycobacterium smegmatis porin A (MspA) nanopore. GCE allows for rapid and efficient introduction of bioorthogonal reactive site (i.e., azide) to the pore rim, and conjugation of single stranded DNA or lysozyme was demonstrated. The lysozyme-conjugated pore was further used for the discrimination of different oligosaccharides, demonstrating a sensing capacity that a bare MspA nanopore does not possess. GCE with bioorthogonal handles, which has never been previously applied in the preparation of nanopores, is a versatile strategy for pore engineering and may further expand the application scenarios of nanopores.     

Designing logical codon reassignment – Expanding the chemistry in biology

Over the last decade, the ability to genetically encode unnatural amino acids (UAAs) has evolved rapidly. The programmed incorporation of UAAs into recombinant proteins relies on the reassignment or suppression of canonical codons with an amino-acyl tRNA synthetase/tRNA (aaRS/tRNA) pair, selective for the UAA of choice. In order to achieve selective incorporation, the aaRS should be selective for the designed tRNA and UAA over the endogenous amino acids and tRNAs. Enhanced selectivity has been achieved by transferring an aaRS/tRNA pair from another kingdom to the organism of interest, and subsequent aaRS evolution to acquire enhanced selectivity for the desired UAA. Today, over 150 non-canonical amino acids have been incorporated using such methods. This enables the introduction of a large variety of structures into proteins, in organisms ranging from prokaryote, yeast and mammalian cells lines to whole animals, enabling the study of protein function at a level that could not previously be achieved. While most research to date has focused on the suppression of ‘non-sense’ codons, recent developments are beginning to open up the possibility of quadruplet codon decoding and the more selective reassignment of sense codons, offering a potentially powerful tool for incorporating multiple amino acids. Here, we aim to provide a focused review of methods for UAA incorporation with an emphasis in particular on the different tRNA synthetase/tRNA pairs exploited or developed, focusing upon the different UAA structures that have been incorporated and the logic behind the design and future creation of such systems. Our hope is that this will help rationalize the design of systems for incorporation of unexplored unnatural amino acids, as well as novel applications for those already known.     

Chemically-modified nanopores for sensing

Sensing with chemically-modified nanopores is an emerging field that is expected to have major impact on bioanalysis and fundamental understanding of nanoscale chemical interactions down to the single-molecule level. The main strength of nanopore sensing is that it implies the prospect of label-free single-molecule detection by taking advantage of the built-in transport-modulation-based amplification mechanism. At present, fabrication and application of solid-state nanopores are becoming the focus of attention because, compared with their biological counterparts, they offer greater flexibility in terms of shape, size, and surface properties, as well as superior robustness. A breakthrough in label-free nanopore sensing for real-world applications is therefore expected from implementing solid-state nanopores, an area that is still developing. Without claiming comprehensiveness, the focus of this review comprises recent results and trends in nanopore-based sensing (i.e. emerging technologies for fabricating solid-state nanopores, their chemical functionalization, and detection methods for quantitative analysis).     

Continuous stochastic detection of amino Acid enantiomers with a protein nanopore

A stochastic sensing method: Discrimination between enantiomeric amino acids is achieved when the amino acids bind to a Cu(II) complex within a protein nanopore sensor, which provides a chiral environment. The potential of the method is demonstrated by real-time observation of the increase in enantiomeric excess during an enzymatic kinetic resolution.     

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.     

Biosynthetic phage display: a novel protein engineering tool combining chemical and genetic diversity

BACKGROUND: Molecular diversity in nature is developed through a combination of genetic and chemical elements. We have developed a method that permits selective manipulation of both these elements in one protein engineering tool. It combines the ability to introduce non-natural amino acids into a protein using native chemical ligation with exhaustive targeted mutagenesis of the protein via phage-display mutagenesis. RESULTS: A fully functional biosynthetic version of the protease inhibitor eglin c was constructed. The amino-terminal fragment (residues 8-40) was chemically synthesized with a non-natural amino acid at position 25. The remaining carboxy-terminal fragment was expressed as a 30-residue peptide extension of gIIIp or gVIIIp on filamentous phage in a phage-display mutagenesis format. Native chemical ligation was used to couple the two fragments and produced a protein that refolded to its active form. To facilitate the packing of the introduced non-natural amino acid, residues 52 and 54 in the carboxy-terminal fragment were fully randomized by phage-display mutagenesis. Although the majority of the observed solutions for residues 52 and 54 were hydrophobic - complementing the stereochemistry of the introduced non-natural amino acid - a significant number of residues (unexpected because of stereochemical and charge criteria) were observed in these positions. CONCLUSIONS: Peptide synthesis and phage-display mutagenesis can be combined to produce a very powerful protein engineering tool. The physical properties of the environment surrounding the introduced non-natural residue can be selected for by evaluating all possible combinations of amino acid types at a targeted set of sites using phage-display mutagenesis.     

Control of the Conductance of Engineered Protein Nanopores through Concerted Loop Motions

Protein nanopores have attracted much interest for nucleic acid sequencing, chemical sensing, and protein folding at the single molecule level. The outer membrane protein OmpG from E. coli stands out because it forms a nanopore from a single polypeptide chain. This property allows the separate engineering of each of the seven extracellular loops that control access to the pore. The longest of these loops, loop 6, has been recognized as the main gating loop that closes the pore at low pH values and opens it at high pH values. A method was devised to pin each of the loops to the embedding membrane and measure the single‐pore conductances of the resulting constructs. The electrophysiological and complementary NMR measurements show that the pinning of individual loops alters the structure and dynamics of neighboring and distant loops in a correlated fashion. Pinning loop 6 generates a constitutively open pore and patterns of concerted loop motions control access to the OmpG nanopore.     

Translation of genetic information on the ribosome

The information for protein structure that is contained in the base sequence of the nucleic acids is translated on the ribosome into the amino acid sequence. This translation can be divided into chain initiation, chain growth, and chain termination. Several specific protein factors and nucleic acids are involved in each section.—For chain initiation, start complexes are formed from the initiating amino acyl-tRNA, mRNA carrying the start signal, and the small and large subunits of a ribosome. GTP and the initiating factors are also involved in this process.—In chain elongation, one amino acid at a time is transferred, in a reaction cycle, from the linkage with tRNA into a linkage with the polypeptide chain. The amino acid to be incorporated is initially bound to the ribosome as amino acyl-tRNA, a process for which GTP and protein factors are necessary. The subsequent formation of a peptide linkage is catalyzed by the peptidyl transferase of the large ribosomal subunit. The peptidyl-tRNA with its newly added amino acid residue is then transferred from the amino acyl-tRNA acceptor site A to the peptidyl donor site P of the ribosome. This requires another protein factor and cleavage of GTP into GDP and phosphate.–Ghain termination begins as soon as one of the three terminator triplets UAA, UAG, or UGA in the mRNA reaches the ribosome. The mRNA is moving in relation to the ribosome from the 5′ end to the 3′ end. Release of the completed polypeptide chain from the ribosome is dependent on release factors. Before initiation of a new polypeptide chain, the ribosomes dissociate into their subunits.     

Functional Nanopores Enabled with DNA

Membrane-spanning nanopores are used in label-free single-molecule sensing and next-generation portable nucleic acid sequencing, and as powerful research tools in biology, biophysics, and synthetic biology. Naturally occurring protein and peptide pores, as well as synthetic inorganic nanopores, are used in these applications, with their limitations. The structural and functional repertoire of nanopores can be considerably expanded by functionalising existing pores with DNA strands and by creating an entirely new class of nanopores with DNA nanotechnology. This review outlines progress in this area of functional DNA nanopores and outlines developments to open up new applications.     

Protein nanopores with covalently attached molecular adapters

Molecular adapters are crucial for the stochastic sensing of organic analytes with alpha-hemolysin (alphaHL) protein nanopores when direct interactions between analytes and the pore cannot readily be arranged by conventional protein engineering. In our earlier studies, cyclodextrin adapters were lodged noncovalently within the lumen of the alphaHL pore. In the present work, we have realized the controlled covalent attachment of a beta-cyclodextrin (betaCD) adapter in the two possible molecular orientations inside alphaHL pores prepared by genetic engineering. There are two advantages to such a covalent system. First, the adapter cannot dissociate, which means there are no gaps during stochastic detection, a crucial advance for single-molecule exonuclease DNA sequencing where the continuous presence of a molecular adapter will be essential for reading individual nucleotides. Second, the ability to orient the adapter allows analytes to bind through only one of the two entrances to the betaCD cavity. We demonstrate that the covalently attached adapters can be used to alter the ion selectivity of the alphaHL pore, examine binding events at elevated temperatures, and detect analytes with prolonged dwell times.     

Block Copolymer‐Derived Monolithic Polymer Films and Membranes Comprising Self‐Organized Cylindrical Nanopores for Chemical Sensing and Separations

Microphase separation of block copolymers (BCPs) has been extensively studied because it leads to the self‐assembled formation of periodic structures controlled on the scale of tens of nanometers. In particular, BCP‐derived cylindrical microdomains have attracted considerable interest for various applications owing to their well‐defined shapes of uniform and tunable diameters. This focus review highlights recent efforts to apply BCP‐derived monolithic films/membranes comprising cylindrical nanopores for chemical sensing and separations. The nanopores provide confined molecular pathways that exhibit enhanced selectivity based on steric, electrostatic, and chemical interactions, and thus, enable us to design unique electrochemical sensors and highly efficient separation membranes.     

光子晶体的分析化学应用研究新进展

光子晶体具有规则的堆积结构、纳米级别的孔径和新颖的光学性质,在工程、物理、化学以及生物学等研究中均有重要的研究与开发价值。本文从分析化学应用研究角度对其进行了审视,综述了最近5年多以来光子晶体在传感与分离分析方法发展中应用的最新进展。     

A designed branched three-helix bundle protein dimer

The ultimate goals of de novo protein design are the construction of novel tertiary structures and functions. Here is presented the design and synthesis of a uniquely branched three-helix bundle that folds into a well-folded dimeric protein. The branching of this protein was performed by the method of native chemical ligation, which provides a chemoselective and stable amide bond between the unprotected fragments. This ligation strategy was possible by the presented facile preparation of a peptide (43 amino acids) with a specific side chain thioester, which is synthesized by general Fmoc solid phase peptide synthesis. From the presented structural analysis, it is seen that the folded protein is present as a stable and highly helical dimer, thus forming a six-helix bundle. This unique tertiary structure, composed of a dimer of three individual alpha-helices branched together, offers different possibilities for protein engineering, such as metal and cofactor binding sites, as well as for the construction of novel functions.     

A Precise Chemical Strategy To Alter the Receptor Specificity of the Adeno‐Associated Virus

The ability to target the adeno‐associated virus (AAV) to specific types of cells, by altering the cell‐surface receptor it binds, is desirable to generate safe and efficient therapeutic vectors. Chemical attachment of receptor‐targeting agents onto the AAV capsid holds potential to alter its tropism, but is limited by the lack of site specificity of available conjugation strategies. The development of an AAV production platform is reported that enables incorporation of unnatural amino acids (UAAs) into specific sites on the virus capsid. Incorporation of an azido‐UAA enabled site‐specific attachment of a cyclic‐RGD peptide onto the capsid, retargeting the virus to the α_vβ₃ integrin receptors, which are overexpressed in tumor vasculature. Retargeting ability was site‐dependent, underscoring the importance of achieving site‐selective capsid modification. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity.     

Switching the Inhibitor-Enzyme Recognition Profile via Chimeric Carbonic Anhydrase XII

A key part of the optimization of small molecules in pharmaceutical inhibitor development is to vary the molecular design to enhance complementarity of chemical features of the compound with the positioning of amino acids in the active site of a target enzyme. Typically this involves iterations of synthesis, to modify the compound, and biophysical assay, to assess the outcomes. Selective targeting of the anti-cancer carbonic anhydrase isoform XII (CA XII), this process is challenging because the overall fold is very similar across the twelve CA isoforms. To enhance drug development for CA XII we used a reverse engineering approach where mutation of the key six amino acids in the active site of human CA XII into the CA II isoform was performed to provide a protein chimera (chCA XII) which is amenable to structure-based compound optimization. Through determination of structural detail and affinity measurement of the interaction with over 60 compounds we observed that the compounds that bound CA XII more strongly than CA II, switched their preference and bound more strongly to the engineered chimera, chCA XII, based on CA II, but containing the 6 key amino acids from CA XII, behaved as CA XII in its compound recognition profile. The structures of the compounds in the chimeric active site also resembled those determined for complexes with CA XII, hence validating this protein engineering approach in the development of new inhibitors.     

Structural information of mussel adhesive protein Mefp-3 acquired at various polymer/Mefp-3 solution interfaces

Mytilus edulis foot protein Mefp-3 serves as a primer in the formation of adhesive plaques that attach the mussel to solid surfaces in its immediate environment. The adsorption behavior of this protein on various materials of different hydrophobicity was studied using sum frequency generation (SFG) vibrational spectroscopy. By collecting SFG signals from side chains of these amino acids and from secondary structures of the protein, we have determined that this protein adopts different conformations at different interfaces, depending on hydrophobicity of the contact medium and specific chemical group interactions. We have also demonstrated that SFG has the potential to track the interfacial conformations of a single amino acid in a protein.     

Protein Organic Chemistry and Applications for Labeling and Engineering in Live‐Cell Systems

The modification of proteins with synthetic probes is a powerful means of elucidating and engineering the functions of proteins both in vitro and in live cells or in vivo. Herein we review recent progress in chemistry‐based protein modification methods and their application in protein engineering, with particular emphasis on the following four strategies: 1) the bioconjugation reactions of amino acids on the surfaces of natural proteins, mainly applied in test‐tube settings; 2) the bioorthogonal reactions of proteins with non‐natural functional groups; 3) the coupling of recognition and reactive sites using an enzyme or short peptide tag–probe pair for labeling natural amino acids; and 4) ligand‐directed labeling chemistries for the selective labeling of endogenous proteins in living systems. Overall, these techniques represent a useful set of tools for application in chemical biology, with the methods 2–4 in particular being applicable to crude (living) habitats. Although still in its infancy, the use of organic chemistry for the manipulation of endogenous proteins, with subsequent applications in living systems, represents a worthy challenge for many chemists.     

Sequence-dependent correction of random coil NMR chemical shifts.

Random coil chemical shifts are commonly used to detect secondary structure elements in proteins in chemical shift index calculations. While this technique is very reliable for folded proteins, application to unfolded proteins reveals significant deviations from measured random coil shifts for certain nuclei. While some of these deviations can be ascribed to residual structure in the unfolded protein, others are clearly caused by local sequence effects. In particular, the amide nitrogen, amide proton, and carbonyl carbon chemical shifts are highly sensitive to the local amino acid sequence. We present a detailed, quantitative analysis of the effect of the 20 naturally occurring amino acids on the random coil shifts of (15)N(H), (1)H(N), and (13)CO resonances of neighboring residues, utilizing complete resonance assignments for a set of five-residue peptides Ac-G-G-X-G-G-NH(2). The work includes a validation of the concepts used to derive sequence-dependent correction factors for random coil chemical shifts, and a comprehensive tabulation of sequence-dependent correction factors that can be applied for amino acids up to two residues from a given position. This new set of correction factors will have important applications to folded proteins as well as to short, unstructured peptides and unfolded proteins.     

Engineered Biocatalytic Synthesis of β-N-Substituted-α-Amino Acids

Non-canonical amino acids (ncAAs) are useful synthons for the development of new medicines, materials, and probes for bioactivity. Recently, enzyme engineering has been leveraged to produce a suite of highly active enzymes for the synthesis of β-substituted amino acids. However, there are few examples of biocatalytic N-substitution reactions to make α,β-diamino acids. In this study, we used directed evolution to engineer the β-subunit of tryptophan synthase, TrpB, for improved activity with diverse amine nucleophiles. Mechanistic analysis shows that high yields are hindered by product re-entry into the catalytic cycle and subsequent decomposition. Additional equivalents of l -serine can inhibit product reentry through kinetic competition, facilitating preparative scale synthesis. We show β-substitution with a dozen aryl amine nucleophiles, including demonstration on a gram scale. These transformations yield an underexplored class of amino acids that can serve as unique building blocks for chemical biology and medicinal chemistry.