人工金属酶分子设计新进展:肌红蛋白研究实例分析

金属酶在生物体中发挥着多种至关重要的作用,而人工金属酶的分子设计能够调控和拓展天然金属酶的功能,甚至创造出功能更为优越的新型酶分子。肌红蛋白（Mb）是作为血红素蛋白或其他金属蛋白分子设计的理想蛋白模型。近些年,基于Mb蛋白骨架的人工金属酶的分子设计逐渐发展了多种研究策略,包括设计氢键网络、金属结合位点、分子内二硫键、利用蛋白翻译后修饰、引入非天然氨基酸和非天然辅基等。本文着重综述这些方面的最新研究进展,可以帮助我们深刻认识金属酶的结构与功能关系,同时掌握人工金属酶分子设计的思路与方法,从而有助于推动这一领域的快速发展。     

蛋白质分子理性设计与肌红蛋白功能的调控及拓展

蛋白质分子理性设计,不但对于揭示天然金属蛋白的结构与功能关系,而且对于创造具有性质和功能改善的人工金属蛋白,都是一个功能强大的策略.肌红蛋白(myoglobin,Mb),自然界创造的一种具有多重生物功能的血红素蛋白,已被证明是蛋白质分子理性设计的理想框架.本文主要综述了两个方面,一是对其天然的氧结合与运输、过氧化物酶(peroxidase)和亚硝酸盐还原酶(nitrite reductase,NIR)的功能进行调控,二是将其功能理性拓展到过氧酶(peroxygenase)、血红素-铜氧化酶(heme-copper oxidase,HCO)、一氧化氮还原酶(nitric oxide reductase,NOR)和羟胺还原酶(hydroxylamine reductase)等.这些研究加深了我们对自然界金属蛋白如何工作的理解,也为将来具有实际应用前景的功能金属蛋白的理性分子设计提供了线索.     

Hb-CTAB/GC电极的电化学行为研究及对NO的电催化检测

生物大分子的直接电化学研究是近年来研究的一个热点[1-3]。血红蛋白(Hb)是一种含血红素辅基的球型蛋白质,在生物体内起存储和运输氧气的作用,其所含的血红素辅基能发生得失电子的反应。但是,在血红蛋白分子中,一方面,血红素辅基被多肽链所包裹,使得血红素辅基不易接近普通电极     

细胞色素c突变研究进展

在简述细胞色素c的生物功能和结构特征的基础上,综述了细胞色素c突变研究的进展,重点论述了对血红素辅基(heme)的轴向配体Met80、heme所在腔的保守氨基酸残基Tyr67以及蛋白表面的保守氨基酸残基Phe82的突变研究,并对一些突变体蛋白表现出来的特殊性质给予解释。     

基于真菌14α-去甲基化酶的非氮唑类抗真菌先导化合物的设计、合成及其体外抗真菌活性研究

基于真菌羊毛甾醇14α-去甲基化酶(CYP51)活性位点的三维结构设计并合成了新型四氢异喹啉类抗真菌先导化合物. 体外抗真菌活性研究显示: 设计的先导化合物具有较好的抗真菌活性. 其中化合物5f和5g对于5种测试菌的抗真菌活性强于或相当于对照药物氟康唑. 先导分子通过与靶酶活性腔氨基酸残基的非共价键结合产生抗真菌作用, 避免与血红素辅基Fe原子发生配位结合, 为一类具有新作用机理的非氮唑类抗真菌先导化合物. 本研究为抗真菌药物研究提供了新的结合方式及结构类型.     

蛋白质功能的扩展与人工金属结合位点的理性设计

生物体系中金属离子在调节金属蛋白的结构和功能中发挥着至关重要的作用。本文综述了利用人工金属结合位点的理性设计来扩展蛋白质功能范围的研究进展,包括在蛋白分子内部通过探索潜在的结合金属位点、重新设计已有的金属结合位点、以及设计全新的金属结合位点的方法来设计人工金属结合位点,和在蛋白分子表面进行设计,来获得结构及功能的转化、研究与纳米材料间的相互作用、以及进行蛋白质分子的自组装。这些研究进展极大地丰富了我们对金属蛋白结构与功能关系的认识。同时,也赋予了我们控制及利用感兴趣蛋白的能力。     

细胞色素b5 S64X突变体对蛋白结构及稳定性的影响

为了深入了解细胞色素b₅(Cyt b₅)64位氨基酸残基(Ser64)对血红素辅基微环境及蛋白性质的影响,我们分别对Cyt b₅ Ser64进行了保守性突变(S64T)以及非保守性突变(S64K、S64N和S64H),均为亲水性氨基酸残基。对野生型细胞色素b₅及其突变体蛋白S64X(X：T,K,N或H)的热、酸、盐酸胍变性的稳定性研究表明：4个突变体蛋白的稳定性相对于野生型都大大降低了;CD光谱表明,细胞色素b₅ S64X突变体中的α-螺旋明显减少,芳香性氨基酸残基所处的肽链结构受到了影响;盐酸胍变性荧光光谱表明,Trp22周围的蛋白肽链受到了影响,Trp22暴露于水溶液的程度加大。我们认为Ser64不仅对血红素辅基有稳定作用,同时还对维持蛋白Core 1中的第5个α-螺旋结构有重要的作用,在64位引入其他氨基酸残基影响了第5个α-螺旋的结构,并通过蛋白肽链的相互作用,使得Trp22所在的Core 2结构也受到了较为明显的扰动。     

电喷雾质谱法研究细胞色素Tb5及其F35Y突变体蛋白 肽链和辅基的相互作用

本文通过牛肝线粒体细胞色素Tb5和它的F35Y突变体蛋白相对分子质量的外标法测定,得到细胞色素Tb5全蛋白的相对分子质量为10077.5脱辅基蛋白的相对分子质量为9461.4F35Y突变体蛋白的相对分子质量为10093.6,它的脱辅基蛋白的相对分子质量为9477.5,不同nozzle电压下的电喷雾质谱结果表明,该电压的大小明显影响蛋白肽链与血红素辅基之间的非共价结合,随着电压的降低,全蛋白谱峰的强度逐渐增大,然而,过低的电压导致了Na^+,K^+离子加合峰相对强度的增加,而不利于谱图分析。同时,考察到细胞色素Tb5在甲醇溶液和酸性溶液中的变性行为,因此选择nozzle电压70V,10%的甲醇水溶液和pH=7为得到全蛋白质谱峰的最佳条件。相同实验条件下得到的野生型CytTb5和F35Y突变体全蛋白的质谱峰相比较,其相对丰度有悬殊的差异,表明F35Y突变体蛋白的血红素结合能力明显低于野生型蛋白。通过解离出的Hemeb的分子离子峰进行解析,证明铁仍以三价离子存在于血红素辅基中。     

电喷雾质谱法研究细胞色素Tb5及其F35Y突变体蛋白肽链和辅基的相互作用

本文通过牛肝线粒体细胞色素Tb5和它的F35Y突变体蛋白相对分子质量的外标法测定,得到细胞色素Tb5全蛋白的相对分子质量为10077.5脱辅基蛋白的相对分子质量为9461.4F35Y突变体蛋白的相对分子质量为10093.6,它的脱辅基蛋白的相对分子质量为9477.5,不同nozzle电压下的电喷雾质谱结果表明,该电压的大小明显影响蛋白肽链与血红素辅基之间的非共价结合,随着电压的降低,全蛋白谱峰的强度逐渐增大,然而,过低的电压导致了Na^+,K^+离子加合峰相对强度的增加,而不利于谱图分析。同时,考察到细胞色素Tb5在甲醇溶液和酸性溶液中的变性行为,因此选择nozzle电压70V,10%的甲醇水溶液和pH=7为得到全蛋白质谱峰的最佳条件。相同实验条件下得到的野生型CytTb5和F35Y突变体全蛋白的质谱峰相比较,其相对丰度有悬殊的差异,表明F35Y突变体蛋白的血红素结合能力明显低于野生型蛋白。通过解离出的Hemeb的分子离子峰进行解析,证明铁仍以三价离子存在于血红素辅基中。     

血红素蛋白质微小仿生模型的设计

金属蛋白种类繁多，功能各异，血红素蛋白质是其中非常重要的一类。为了研究其结构。功能之间的关系，人们采用不同的方法设计了多种血红素蛋白质的微小仿生模型。本文介绍了螺旋束血红素蛋白质，螺旋—血红素—螺旋夹心蛋白质的设计和表征。     

Biosynthetic approach to modeling and understanding metalloproteins using unnatural amino acids

Metalloproteins have inspired chemists for many years to synthesize artificial catalysts that mimic native enzymes.As a complementary approach to studying native enzymes or making synthetic models,biosynthetic approach using small and stable proteins to model native enzymes has offered advantages of incorporating non-covalent secondary sphere interactions under physiological conditions.However,most biosynthetic models are restricted to natural amino acids.To overcome this limitation,incorporating unnatural amino acids into the biosynthetic models has shown promises.In this review,we summarize first synthetic,semisynthetic and biological methods of incorporates unnatural amino acids(UAAs)into proteins,followed by progress made in incorporating UAAs into both native metalloproteins and their biosynthetic models to fine-tune functional properties beyond native enzymes or their variants containing natural amino acids,such as reduction potentials of azurin,O_2 reduction rates and percentages of product formation of HCO models in Mb,the rate of radical transport in ribonucleotide reductase(RNR)and the proton and electron transfer pathways in photosystemⅡ(PSⅡ).We also discuss how this endeavour has allowed systematic investigations of precise roles of conserved residues in metalloproteins,such as Metl21 in azurin,Tyr244 that is cross-linked to one of the three His ligands to CuB in HCO,Tyr122,356,730 and 731 in RNR and TyrZ in PSⅡ.These examples have demonstrated that incorporating UAAs has provided a new dimension in our efforts to mimic native enzymes and in providing deeper insights into structural features responsible high enzymatic activity and reaction mechanisms,making it possible to design highly efficient artificial catalysts with similar or even higher activity than native enzymes.     

Toward stereocontrolled, chemoenzymatic synthesis of unnatural peptides

An efficient, chemoenzymatic method for the multicomponent synthesis of unnatural tripeptides is presented. Development of a previously described procedure combines the diversity offered by multicomponent reactions with the selectivity of biocatalysts and allows the convenient introduction of varied amino acid moieties into the tripeptide scaffold, with control of the stereochemistry. Additionally, it allows the introduction of a methyl group to the amide nitrogen, leading to derivatives of N-methylated amino acids.     

Fluorinated amino acids in protein design and engineering

Selective incorporation of unnatural amino acids into proteins is a powerful tool for illuminating the principles of protein design. In particular, fluorinated amino acids have recently emerged as valuable building blocks for designing hyperstable protein folds, as well as directing highly specific protein-protein interactions. We review the collagen mimetic and coiled coil peptide systems that exemplify generalizable paradigms for future design. The unique electronic and phase properties of fluorocarbons are discussed, and protein synthesis using unnatural amino acids is briefly reviewed.     

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.     

Palladium‐Catalyzed Asymmetric Benzylation of Azlactones

Asymmetric benzylation of prochiral azlactone nucleophiles enables the catalytic introduction of a benzyl group towards the synthesis of α,α‐disubstituted amino acids. Herein, we report an enantioselective palladium‐catalyzed process using chiral bis(diphenylphosphinobenzoyl)diamine (dppba) ligands. Naphthalene‐ and heterocycle‐based methyl carbonates react with a number of azlactones derived from both natural and unnatural amino acids. Monocyclic benzylic electrophiles, for which the barrier to ionization is higher, must employ a phosphate leaving group in order to react. Reaction conditions for electron‐rich and ‐neutral benzylic electrophiles have been developed, and the scope of the reaction has been explored with respect to both reaction partners. The high levels of asymmetric induction, as well as the reactivity pattern of the electrophiles, suggest an η³‐benzyl intermediate that arises through two distinct pathways.     

Ribosomal synthesis of unnatural peptides

Combinatorial libraries of non-biological polymers and drug-like peptides could in principle be synthesized from unnatural amino acids by exploiting the broad substrate specificity of the ribosome. The ribosomal synthesis of such libraries would allow rare functional molecules to be identified using technologies developed for the in vitro selection of peptides and proteins. Here, we use a reconstituted E. coli translation system to simultaneously re-assign 35 of the 61 sense codons to 12 unnatural amino acid analogues. This reprogrammed genetic code was used to direct the synthesis of a single peptide containing 10 different unnatural amino acids. This system is compatible with mRNA-display, enabling the synthesis of unnatural peptide libraries of 10(14) unique members for the in vitro selection of functional unnatural molecules. We also show that the chemical space sampled by these libraries can be expanded using mutant aminoacyl-tRNA synthetases for the incorporation of additional unnatural amino acids or by the specific posttranslational chemical derivitization of reactive groups with small molecules. This system represents a first step toward a platform for the synthesis by enzymatic tRNA aminoacylation and ribosomal translation of cyclic peptides comprised of unnatural amino acids that are similar to the nonribosomal peptides.     

Conformationally rigid aromatic amino acids as potential building blocks for abiotic foldamers

This communication describes the development of conformationally constrained unnatural aromatic amino acids, constructed on rigid backbone wherein the carboxyl and amino groups project in two dimensions (planes) on the aromatic framework. Such a feature offers the possibility of design and development of conformationally ordered synthetic oligomers with intriguing structural architectures distinct from those classically observed. Furthermore, such amino acids will have the potential to extend the conformational space available for foldamer design with diverse backbone conformation and structural architectures.     

Reprogramming the genetic code: from triplet to quadruplet codes

The genetic code of cells is near-universally triplet, and since many ribosomal mutations are lethal, changing the cellular ribosome to read nontriplet codes is challenging. Herein we review work on the incorporation of unnatural amino acids into proteins in response to quadruplet codons, and the creation of an orthogonal translation system in the cell that uses an evolved orthogonal ribosome to efficiently direct the incorporation of unnatural amino acids in response to quadruplet codons. Using this system multiple distinct unnatural amino acids have been incorporated and used to genetically program emergent properties into recombinant proteins. Extension of approaches to incorporate multiple unnatural amino acids may allow the combinatorial biosynthesis of materials and therapeutics, and drive investigations into whether life with additional genetically encoded polymers can evolve to perform functions that natural biological systems cannot.     

Benzophenone Schiff bases of glycine derivatives: Versatile starting materials for the synthesis of amino acids and their derivatives

This review focuses on the introduction and early development, in solution, of phase-transfer catalyzed (PTC) reactions to afford racemic or enantioenriched natural and unnatural amino acids. To form monosubstituted amino acids alkylation reactions are performed on the benzophenone Schiff base of glycine. For α,α-disubstituted amino acids the activated intermediate is an aldimine derivative of the monosubstituted amino acid. Enantioenriched products are produced by organocatalysis using derivatives of Cinchona alkaloids as the phase-transfer catalyst. Selectivity for monoalkylatation and lack of product racemization depend on the acidities of the glycine imines, and dialkylated products are formed from aldimine esters of monoalkyl amino acids. The racemic and catalytic enantioselective reactions of a cationic glycine equivalent with organoboranes, organometallics and malonate anion are discussed as are other reactions of these versatile Schiff bases derivatives.     

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.