Proteins with β-(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids

L-beta-(Thieno[3,2-b]pyrrolyl)alanine and L-beta-(thieno[2,3-b]pyrrolyl)alanine are mutually isosteric and pharmaceutically active amino acids that mimic tryptophan with the benzene ring in the indole moiety replaced by thiophene. Sulfur as a heteroatom causes physicochemical changes in these tryptophan surrogates that bring about completely new properties not found in the indole moiety. These synthetic amino acids were incorporated into recombinant proteins in response to the Trp UGG codons by fermentation in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. Related protein mutants expectedly retain the secondary structure of the native proteins but show significantly changed optical and thermodynamic properties. In this way, new spectral windows, fluorescence, polarity, thermodynamics, or pharmacological properties are inserted into proteins. Such an engineering approach by translational integration of synthetic amino acids with a priori defined properties, as shown in this study, proved to be a novel and useful tool for protein rational design.     

Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code

The expanded genetic code in combination with site-directed mutagenesis was used to probe spectroscopic and structural roles of tryptophan (Trp) residues in Aequorea victoria green fluorescent proteins (avGFPs). Nine different halogen-, chalcogen-, and methyl-containing Trp isosteric analogues and surrogates were incorporated into avGFPs containing indole moieties in, and outside of, the chromophore, by the use of the selective pressure incorporation method. Such isosteric replacements introduced minimal local geometry changes in indole moieties, often to the level of single atomic exchange ('atomic mutation') and do not affect three-dimensional structures of avGFPs but induce changes in spectral properties. Our approach offers a new platform to re-evaluate issues like resonance transfer, mechanisms of chromophore formation and maturation, as well as the importance of local geometry and weak sulphur-aromatic interactions for avGFP spectral properties and structural stability. The library of novel tailor-made avGFP mutants and variants generated in this work has demonstrated not only the potentials of the expanded genetic code to study spectroscopic functions, but also a new approach to generate tailor-made proteins with interesting and useful spectral properties.     

In vivo engineering of proteins with nitrogen-containing tryptophan analogs

Recently, it has become possible to reprogram the protein synthesis machinery such that numerous noncanonical amino acids can be translated into target sequences yielding tailor-made proteins. The canonical amino acid tryptophan (Trp) encoded by a single nucleotide triplet (UGG) is a particularly interesting target for protein engineering and design. Trp-residues can be substituted with a variety of analogs and surrogates generated biosynthetically or by organic chemistry. Among them, nitrogen-containing tryptophan analogs occupy a central position, as they have distinct chemical properties in comparison with aliphatic amines and imines. They resemble purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. These special properties of the analogs can be directly transmitted into related protein structures via in vivo ribosome-mediated translation. Proteins expressed in this way are further endowed with unique properties like new spectral, altered redox and titration features or might serve as useful biomaterials. We present and discuss current works and future developments in protein engineering with nitrogen-containing tryptophan analogs and related compounds as well as their relevance for academic and applicative research.The term noncanonical amino acid refers to an amino acid that does not belong, in contrast to a canonical amino acid, to the genetically encoded, proteinogenic amino acids. The term analog defines a strict isosteric exchange of a canonical/noncanonical amino acid (e.g., tryptophan/azatryptophan), while the term surrogate defines a nonisosteric change (e.g., tryptophan/azulene). Mutant denotes a protein in which the wild-type sequence was changed by site-directed mutagenesis (codon manipulation on the DNA level) within the repertoire of the standard amino acids. Variant denotes a protein in which one or more canonical amino acids derived from a wild-type or a mutant sequence were replaced by a noncanonical one (expanded amino acid repertoire, codon reassignment on the protein translation level).     

Protein-protein interaction using tryptophan analogues: novel spectroscopic probes for toxin-elongation factor-2 interactions

Previously, we characterized the role of the three naturally occurring Trp residues (W-417, -466, and -558) in the catalytic mechanism of the toxin-enzyme produced by Pseudomonas aeruginosa [Beattie and Merrill (1999) J. Biol. Chem. 274, 15646-15654]. However, the use of intrinsic Trp fluorescence to study toxin-eEF-2 interaction is inherently limited since the spectral properties of the various Trp residues in both proteins cannot easily be distinguished. To facilitate the study of the protein-protein interaction by Trp fluorescence spectroscopy, the Trp residues in the catalytic domain of exotoxin A were replaced with the amino acid analogues 4-fluorotryptophan, 5-fluorotryptophan, 5-hydroxytryptophan, and 7-azatryptophan. The incorporation of analogues was achieved by using a tightly regulated promoter, pBAD, and expressing the protein in a Trp auxotrophic strain of Escherichia coli, BL21, in a minimal medium containing the appropriate tryptophan analogue. Quantitative spectral analysis of the analogue-containing proteins using the Decompose program indicated that we had achieved 87-100% incorporation efficiency depending on the Trp analogue being used. Electrospray mass spectrometry analysis verified that we had achieved nearly total replacement of the L-tryptophan residues within the catalytic domain of exotoxin A with the tryptophan analogues 5-fluorotryptophan and 4-fluorotryptophan. The analogue-substituted proteins showed a variation in their catalytic activities with k(cat) values ranging from 6-fold (4-fluorotryptophan) to 260-fold (5-hydroxytryptophan) lower than the natural enzyme, which was in agreement with previous data using site-directed mutagenesis [Beattie et al. (1996) Biochemistry 35, 15134-15142]. However, the analogue-incorporated enzymes did not show any significant change in their ability to bind NAD(+) as substrate, as determined from a fluorescence-binding assay. The spectral properties of the various analogue-incorporated proteins were evaluated and compared with those of the native protein. Furthermore, selective excitation of the 5-hydroxytryptophan-incorporated toxin was exploited to study its interaction with the elongation factor-2 substrate by fluorescence resonance energy transfer to an acceptor chromophore located on the elongation factor-2 protein. The binding between the toxin-enzyme and elongation factor-2 was shown to be independent of the NAD(+) substrate (983 +/- 63 nM) and showed a small dependence upon the ionic strength of the solution.     

Aminotryptophan-containing barstar: structure--function tradeoff in protein design and engineering with an expanded genetic code

The indole ring of the canonical amino acid tryptophan (Trp) possesses distinguished features, such as sterical bulk, hydrophobicity and the nitrogen atom which is capable of acting as a hydrogen bond donor. The introduction of an amino group into the indole moiety of Trp yields the structural analogs 4-aminotryptophan ((4-NH(2))Trp) and 5-aminotryptophan ((5-NH(2))Trp). Their hydrophobicity and spectral properties are substantially different when compared to those of Trp. They resemble the purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. The Trp --> aminotryptophan substitution in proteins during ribosomal translation is expected to result in related protein variants that acquire these features. These expectations have been fulfilled by incorporating (4-NH(2))Trp and (5-NH(2))Trp into barstar, an intracellular inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens. The crystal structure of (4-NH(2))Trp-barstar is similar to that of the parent protein, whereas its spectral and thermodynamic behavior is found to be remarkably different. The T(m) value of (4-NH(2))Trp- and (5-NH(2))Trp-barstar is lowered by about 20 degrees Celsius, and they exhibit a strongly reduced unfolding cooperativity and substantial loss of free energy in folding. Furthermore, folding kinetic study of (4-NH(2))Trp-barstar revealed that the denatured state is even preferred over native one. The combination of structural and thermodynamic analyses clearly shows how structures of substituted barstar display a typical structure-function tradeoff: the acquirement of unique pH-sensitive charge transfer as a novel function is achieved at the expense of protein stability. These findings provide a new insight into the evolution of the amino acid repertoire of the universal genetic code and highlight possible problems regarding protein engineering and design by using an expanded genetic code.     

Structural study of melanocortin peptides by fluorescence spectroscopy: identification of beta-(2-naphthyl)-D-alanine as a fluorescent probe

Several cyclic disulfide alpha-melanocyte stimulating hormone (alpha-MSH) analogues containing the aromatic fluorescent amino acid beta-(2-naphthyl)-D-alanine (D-Nal) have high affinity and selectivity for the melanocortin (MC)-4 receptor. Considering the possible relevant role played by the lipid phase in the peptide-receptor interaction, the structures of two cyclic alpha-MSH analogues, containing both Trp and D-Nal fluorophores, were investigated by steady-state and time-resolved fluorescence spectroscopy, in aqueous solution and in the presence of dimyristoyl phosphatidylglycerol (DMPG) vesicles, and compared with that of the natural peptide. The amino acid D-Nal gives a unique de-excitation fluorescence profile, with an excited state lifetime much longer than those of Trp, allowing good distinction between the two fluorophores. The cyclic analogues' aqueous structures seem to be adequate for membrane penetration, as Trp fluorescence indicates that, in both aqueous and lipid media, the Trp environment in the cyclic peptides is similar to that of alpha-MSH when incorporated in lipid bilayers. Trp, in the cyclic analogues, seems to penetrate deeper in the bilayer than in the native peptide. The amino acid D-Nal was also found to penetrate deep into the lipid bilayer, having its excited-state lifetime drastically changed from aqueous solution to lipid medium. The present work shows that D-Nal may serve as a fluorescent probe for studies of MC peptides and suggests that the high affinity and selectivity of the cyclic peptides to the MC4 membrane receptor could be related to their deeper penetration into the bilayer core.     

Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog

Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.     

A new approach to gene mutation analysis using "GFP-Display"

The unique behavior of green fluorescent protein (GFP) on SDS-PAGE was applied to the detection of a single amino acid substitution in GFP-tagged polypeptides. This simple detection method using SDS/urea gels was designated GFP-display. The N-terminal 18 or 37 amino acids of K-Ras was used as a model GFP-tagged polypeptide. K-ras exon 1 was fused to a gfp cDNA at each end and expressed in Escherichia coli. Amino acid number 12 of K-Ras (wild type; Gly) was changed to Ser, Arg, Cys, Asp, Ala, or Val, and the mobility shift of the greenish fluorescent bands in the SDS/urea gel was analyzed. These mutants were easily detected by GFP-display; however, detection depended strongly on the urea concentration and electrophoresis temperature. Subsequently, GFP-display was applied to the 36 amino acids encoding human p53 exon 7. Amino acid number 248 (wild type; Arg) was changed to Gly, Trp, Gln, Pro, or Leu, and similar mobility shifts were observed. GFP-display could be coupled with an in vitro translation system. Fluorescent active GFP and GFP-Ras fusion proteins were synthesized within a few hours. GFP-display shows potential as a modern approach to gene mutation analysis at the protein level, and is a useful method for protein engineering studies.     

Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini

N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein in vivo and demonstrate it by preparing green fluorescent protein (GFP) having bio-orthogonally reactive groups at its N-terminus. In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L-homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N-terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces.     

Protein and cellular engineering with unnatural amino acids

Proteins are the central functional constituents in all living organisms ranging from viruses, bacteria, yeast, and plants to mammals. All of these biopolymers that are formed by natural biosynthetic pathways are composed of a genetically determined sequence of the 20 so-called natural amino acids. The physical and chemical properties of proteins are a reflection of the side chains of each of the component amino acids. However, for some purposes it would be very desireable to have amino acids with side chains of various selected physical chemical properties, such as a keto group, a crosslinker, or a NMR probe group, incorporated into the protein. Although chemical and biochemical methods for modifying amino acid moieties in proteins have been achieved, recent successes in incorporating unnatural amino acids in vivo open entirely new avenues for determining protein functions in vivo and for the creation of unnatural proteins with novel functionalities. Several examples by employing the novel activity of unnatural amino acids have shown significant roles in both basic research and biotechnology.     

Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo.

Many proteins with unusual structural properties are comprised of multiple repeating amino acid sequences and are often fractious to expression in recombinant systems. To facilitate recombinant production of such proteins for structural and engineering studies, we have produced circular messenger RNAs with infinite open reading frames. We show that a circular mRNA containing a simple green fluorescent protein (GFP) open reading frame can direct GFP expression in Escherichia coli. A circular mRNA with an infinite GFP open reading frame produces extremely long protein chains, proving that bacterial ribosomes can internally initiate and repeatedly transit a circular mRNA. Only the monomeric forms of GFP produced from circular mRNA are fluorescent. Analysis of the translation initiation region shows that multiple sequences contribute to maximal translation from circular mRNA. This technology provides a unique means of producing a very long repeating-sequence protein, and may open the way for development of proteinaceous materials with novel properties.     

Substrate-Induced Conformational Changes of the Tyrocidine Synthetase 1 Adenylation Domain Probed by Intrinsic Trp Fluorescence

Nonribosomal peptide synthetases (NRPS) are multifunctional proteins that catalyze the synthesis of the peptide products with enormous biological potential. The process of biosynthesis starts with the adenylation (A) domain, which during the catalytic cycle undergoes extensive structural rearrangements. In this paper, we present the first study of the tyrocidine synthetase 1 A-domain (TycA-A) fluorescence properties. The TycA-A protein contains five potentially fluorescent Trp residues at positions 227, 301, 323, 376 and 406. The contribution of each Trp to the TycA-A emission was determined using protein variants bearing single Trp to Phe substitutions. The accessibility of the Trp side chains during adenylation showed that only W227 is affected by substrate binding. The protein variant containing solely fluorescent W227 residue was constructed and further used as a probe to explore the binding effect of different non-cognate amino acid substrates. The results indicate a different accessibility of W227 residue in the presence of non-cognate amino acids, which might offer an explanation for the higher aminoacyl-adenenylate leakage. Overall, our results suggest that intrinsic tryptophan fluorescence could be used as a method to probe the effect of substrate binding on the local structure in NRPS adenylation domains.     

Expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins

Much effort has been dedicated to the design of significantly red shifted variants of the green fluorescent protein (GFP) from Aequoria victora (av). These approaches have been based on classical engineering with the 20 canonical amino acids. We report here an expansion of these efforts by incorporation of an amino substituted variant of tryptophan into the "cyan" GFP mutant, which turned it into a "gold" variant. This variant possesses a red shift in emission unprecedented for any avFP, similar to "red" FPs, but with enhanced stability and a very low aggregation tendency. An increasing number of non-natural amino acids are available for chromophore redesign (by engineering of the genetic code) and enable new general strategies to generate novel classes of tailor-made GFP proteins.     

Efforts towards the design of 'teflon' proteins: in vivo translation with trifluorinated leucine and methionine analogues

In vivo incorporation of monofluorinated noncanonical amino acids into recombinant proteins has been well-established for decades. Proteins fluorinated in this way proved to be useful tools for many practical applications. In contrast, trifluorinated amino acids have been incorporated in only a few peptides and relatively small proteins by using expression systems in living cells. A novel class of proteins with a fluorous core can be envisaged only if full replacement of the core-building hydrophobic and aliphatic amino acids such as leucine or methionine with the related analogues trifluoromethionine and trifluoroleucine would be feasible. However, our systematic efforts to introduce these amino acids in larger proteins (over 10 Da) that contain different structural motifs clearly show that only partial substitutions are possible. The reasons are high toxicity of these substances and difficulties to accommodate them into the compact cores of natural proteins without adverse effects on their structural integrity. Therefore, engineering of such three dimensional 'Teflon'-like structures would require, besides an expansion of the amino acid repertoire of the genetic code, a de novo protein design as well.     

无细胞体系非天然蛋白质合成研究进展

无细胞非天然蛋白质合成作为蛋白质研究的新兴手段,已成功用于表征蛋白质分子间、蛋白质与核酸分子间相互作用等基础科学研究及医药蛋白、蛋白质材料等工业生产领域。无细胞非天然蛋白质合成系统不需维持细胞的生长,无细胞膜阻碍,可依据研究目的添加基因元件或化学物质从而增强工程设计和过程调控的自由性;也可赋予蛋白质新的特性、结构及功能,如可实现蛋白翻译后修饰、反应手柄引入、生物物理探针及多聚蛋白质合成等。文中系统地综述了目前应用于无细胞蛋白质合成系统中的非天然氨基酸嵌入方法,包括全局抑制及基于正交翻译体系的终止密码子抑制、移码抑制、有义密码子再分配和非天然碱基等方法的研究进展,及非天然氨基酸在蛋白质修饰、生物物理探针、酶工程、蛋白质材料以及医药蛋白质生产等领域的应用进展,并分析了该体系的发展前景及广泛工业化应用的机遇与挑战。     

Partitioning of peptides and recombinant protein–peptide fusions in thermoseparating aqueous two-phase systems: effect of peptide primary structure

Genetic engineering has been used for fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in an aqueous two-phase system. The system was composed of dextran and the thermoseparating ethylene oxide–propylene oxide random copolymer, EO30PO70. Peptides containing tryptophan, proline, arginine or aspartate residues were fused at the C-terminus of the recombinant protein ZZ-cutinase. The aim was to find effective tags for the lipolytic enzyme cutinase for large-scale extraction. The target protein and peptide tags were partitioned separately and then together in the fusion proteins in order to gain increased understanding of the influence of certain amino acid residues on the partitioning. The salt K₂SO₄ was used to reduce the charge dependent salt effects on partitioning and to evaluate the contribution to the partition coefficient from the hydrophobic–hydrophilic properties of the amino acid residues. The effect of Trp on peptide partitioning was independent of the difference in primary structure for (Trp)n, (Trp-Pro)n, (Ala-Trp-Trp-Pro)n and was only determined by the number of Trp. The effect of the charged residues, Arg and Asp, was dependent on the surrounding residues, i.e. if they were situated next to Trp or not. The partitioning behaviour observed for the peptides was qualitatively and in some cases also quantitatively the same as for the fusion proteins. The effect of the salts sodium perchlorate and triethylammonium phosphate on the partitioning was also studied. The salt effects observed for the peptides were qualitatively similar to the effects observed for the fusion proteins.     

Novel biosensor system model based on fluorescence quenching by a fluorescent streptavidin and carbazole‐labeled biotin

In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole‐labeled biotin. BODIPY‐FL‐aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position‐specifically incorporated into Trp120 position of streptavidin by four‐base codon method. On the other hand, carbazole‐labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole‐labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole‐labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole‐labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd.     

Mapping proximity within proteins using fluorescence spectroscopy. A study of T4 lysozyme showing that tryptophan residues quench bimane fluorescence

We present a novel method for mapping proximity within proteins. The method exploits the quenching of the fluorescent label bimane by nearby Trp residues. In studies of T4 lysozyme we show that this effect appears to be distance dependent and orientation specific. Specifically, we show that a proximal Trp residue can reduce bimane fluorescence intensity by up to 500% and induce complicated fluorescence decay kinetics. Replacing the neighboring Trp residue with phenylalanine removes these spectral perturbations. The advantages of using the Trp quenching of bimane fluorescence for protein structural studies include the low amount of protein required and the substantial simplification of labeling strategies. We anticipate this method will prove suitable for a wide array of high-throughput protein studies such as protein folding, the detection of protein-protein interactions, and, most importantly, the dynamic monitoring of conformational changes.     

利用串联质谱鉴定氨基酸突变的生物信息学算法

氨基酸突变能够改变蛋白的结构和功能,影响生物体的生命过程.基于串联质谱的鸟枪法蛋白质组学是目前大规模研究蛋白质组学的主要方法,但是现有的质谱数据鉴定流程为了提高鉴定结果的灵敏度往往会有意压缩数据库中的氨基酸突变信息.因此,如何挖掘数据中的氨基酸突变信息成为当前质谱数据鉴定的一个重要部分.当前应用于氨基酸突变鉴定的串联质谱鉴定方法大致可以分为3大类:基于序列数据库搜索的方法、基于序列标签搜索的算法以及基于图谱库搜索的算法.本文首先详细介绍了这3种氨基酸突变鉴定算法,并分析了各种方法的特点和不足,然后介绍了氨基酸突变鉴定的研究现状和发展方向.随着基于串联质谱的蛋白质组学的不断发展,蛋白序列中的氨基酸突变信息将被更好地解析出来,从而得以深入探讨由氨基酸突变引起的蛋白结构和功能改变,为揭示氨基酸突变的生物学意义奠定基础.     

Photomodulatable fluorescent proteins for imaging cell dynamics and cell fate

An organism arises from the coordinate generation of different cell types and the stereotypical organization of these cells into tissues and organs. Even so, the dynamic behaviors, as well as the ultimate fates, of cells driving the morphogenesis of an organism, or even an individual organ, remain largely unknown. Continued innovations in optical imaging modalities, along with the discovery and evolution of improved genetically-encoded fluorescent protein reporters in combination with model organism, stem cell and tissue engineering paradigms are providing the means to investigate these unresolved questions. The emergence of fluorescent proteins whose spectral properties can be photomodulated is one of the most significant new developments in the field of cell biology where they are primarily used for studying protein dynamics in cells. Likewise, the use of photomodulatable fluorescent proteins holds great promise for use in developmental biology. Photomodulatable fluorescent proteins also represent attractive and emergent tools for studying cell dynamics in complex populations by facilitating the labeling and tracking of individual or defined groups of cells. Here, we review the currently available photomodulatable fluorescent proteins and their application in model organisms. We also discuss prospects for their use in mice, and by extension in embryonic stem cell and tissue engineering paradigms.