Utilization of sterol carrier protein-2 by phytanoyl-CoA 2-hydroxylase in the peroxisomal alpha oxidation of phytanic acid

Since it possesses a 3-methyl group, phytanic acid is degraded by a peroxisomal alpha-oxidation pathway, the first step of which is catalyzed by phytanoyl-CoA 2-hydroxylase (PAHX). Mutations in human PAHX cause phytanic acid accumulations leading to Adult Refsum's Disease (ARD), which is also observed in a sterol carrier protein 2 (SCP-2)-deficient mouse model. Phytanoyl-CoA is efficiently 2-hydroxylated by PAHX in vitro in the presence of mature SCP-2. Other straight-chain fatty acyl-CoA esters were also 2-hydroxylated and the products isolated and characterized. Use of SCP-2 increases discrimination between straight-chain (e.g., hexadecanoyl-CoA) and branched-chain (e.g., phytanoyl-CoA) substrates by PAHX. The results explain the phytanic acid accumulation in the SCP-2-deficient mouse model and suggest that some of the common symptoms of ARD and other peroxisomal diseases may arise in part due to defects in SCP-2 function caused by increased phytanic acid levels.     

Halomethyl-Triazoles for Rapid,Site-Selective Protein Modification

Post-translational modifications (PTMs) are used by organisms to control protein structure and function after protein translation, but their study is complicated and their roles are not often well understood as PTMs are difficult to introduce onto proteins selectively. Designing reagents that are both good mimics of PTMs, but also only modify select amino acid residues in proteins is challenging. Frequently, both a chemical warhead and linker are used, creating a product that is a misrepresentation of the natural modification. We have previously shown that biotin-chloromethyl-triazole is an effective reagent for cysteine modification to give S-Lys derivatives where the triazole is a good mimic of natural lysine acylation. Here, we demonstrate both how the reactivity of the alkylating reagents can be increased and how the range of triazole PTM mimics can be expanded. These new iodomethyl-triazole reagents are able to modify a cysteine residue on a histone protein with excellent selectivity in 30 min to give PTM mimics of acylated lysine side-chains. Studies on the more complicated, folded protein SCP-2L showed promising reactivity, but also suggested the halomethyl-triazoles are potent alkylators of methionine residues.     

Novel chemometric strategy based on the application of artificial neural networks to crossed mixture design for the improvement of recombinant protein production in continuous culture

The optimal blends of six compounds that should be present in culture media used in recombinant protein production were determined by means of artificial neural networks (ANN) coupled with crossed mixture experimental design. This combination constitutes a novel approach to develop a medium for cultivating genetically engineered mammalian cells. The compounds were collected in two mixtures of three elements each, and the experimental space was determined by a crossed mixture design. Empirical data from 51 experimental units were used in a multiresponse analysis to train artificial neural networks which satisfy different requirements, in order to define two new culture media (Medium 1 and Medium 2) to be used in a continuous biopharmaceutical production process. These media were tested in a bioreactor to produce a recombinant protein in CHO cells. Remarkably, for both predicted media all responses satisfied the predefined goals pursued during the analysis, except in the case of the specific growth rate (μ) observed for Medium 1. ANN analysis proved to be a suitable methodology to be used when dealing with complex experimental designs, as frequently occurs in the optimization of production processes in the biotechnology area. The present work is a new example of the use of ANN for the resolution of a complex, real life system, successfully employed in the context of a biopharmaceutical production process.     

Computational Design and Study of Artificial Selenoenzyme with Controllable Activity Based on an Allosteric Protein Scaffold

The establishment of new enzymatic function in an existing scaffold is a great challenge for protein engineers. In previous work, a highly efficient artificial selenoenzyme with controllable activity was constructed, based on a Ca²⁺-responsive recoverin (Rn) protein. In this study, a design strategy combining docking, molecular dynamics, and MM-PBSA is presented, to predict the catalytically active site of glutathione peroxidase (GPx) on the allosteric domain of Rn. The energy contributions of the binding hot spot residues are evaluated further by energy decomposition analysis to determine the detailed substrate recognition mechanism of Rn, which provides clear guidance for artificial enzyme design for improved substrate binding (Michaelis–Menten constant, K_m).     

A triply templated artificial beta-sheet.

This paper describes the design, synthesis, and structural evaluation of a compound (4) comprising three molecular templates and a peptide strand that mimics a three-stranded protein beta-sheet. Two of the templates mimic the hydrogen-bonding functionality of peptide beta-strands and serve as the top and bottom strands by embracing the peptide strand, which is located in the middle of the sheet. The remaining template holds the three strands next to each other. The synthesis of artificial beta-sheet 4 begins with the bottom template and involves the sequential addition of the middle and top strands. (1)H NMR chemical shift and NOE studies establish that this compound folds to adopt a hydrogen-bonded beta-sheetlike structure in CDCl(3) solution. Chemical shift studies indicate that triply stranded artificial beta-sheet 4 is more tightly folded than its smaller doubly stranded homologue, artificial beta-sheet 1.     

Spidroin‐Inspired,High‐Strength,Loofah‐Shaped Protein Fiber for Capturing Uranium from Seawater

The unique three‐dimensional structure of spidrion determines the outstanding mechanical properties of the spider silk fiber. Inspired by the similarity of the three‐dimensional structure of superb‐uranyl binding protein (SUP) to that of spidroin, a dual‐SUP (DSUP) chimeric protein fiber with high tensile strength is designed. The DSUP hydrogel fiber exhibits a loofah‐shape structure by the cross‐interaction of the protein nanofiber. Full exposure of abundant functional uranyl‐binding sites in the stretchable loofah‐shape hydrogel protein fiber give the DSUP fiber a groundbreaking uranium extraction capacity of 17.45 mg g^?1 with an ultrashort saturation time of 3 days in natural seawater. This work reports the design of an adsorbent with ultrahigh uranium extraction capacity and explores a strategy for fabricating artificial high‐strength functional non‐spidroin protein fiber.     

Semi-synthesis of an artificial scandium(iii) enzyme with a β-helical bio-nanotube

We have succeeded in preparing semi-synthesized proteins bound to Sc(3+) ion which can promote an epoxide ring-opening reaction. The Sc(3+) binding site was created on the surface of [(gp5βf)(3)](2) (N. Yokoi et al., Small, 2010, 6, 1873) by introducing a cysteine residue for conjugation of a bpy moiety using a thiol-maleimide coupling reaction. Three cysteine mutants [(gp5βf_X)(3)](2) (X = G18C, L47C, N51C) were prepared to introduce a bpy in different positions because it had been reported that Sc(3+) ion can serve as a Lewis-acid catalyst for an epoxide ring-opening reaction upon binding of epoxide to bpy and two -ROH groups. G18C_bpy with Sc(3+) can accelerate the rate of catalysis of the epoxide ring-opening reaction and has the highest rate of conversion among the three mutants. The value is more than 20 times higher than that of the mixtures of [(gp5βf)(3)](2)/2,2'-bipyridine and l-threonine/2,2'-bipyridine. The elevated activity was obtained by the cooperative effect of stabilizing the Sc(3+) coordination and accumulation of substrates on the protein surface. Thus, we expect that the semi-synthetic approach can provide insights into new rational design of artificial metalloenzymes.     

A ten-residue fragment of an antibody (mini-antibody) directed against lysozyme as ligand in immunoaffinity chromatography

The interaction between an antibody molecule and a protein antigen is an example of "natural" protein modelling. Amino acids of the antigen-binding site consisting of three hypervariable segments (L1, L2, L3) of the light (L) and three (H1, H2, H3) of the heavy (H) chain of an antibody molecule interact with amino acids present in an epitope of a protein. A ten-residue peptide was synthesized with an amino acid sequence analogous to the hypervariable L3 segment of a monoclonal antibody directed against lysozyme. The peptide was immobilized on CH-Sepharose 4B and the affinity adsorbent was used to purify lysozyme added to a detergent extract of insect cells infected with a recombinant baculovirus. This methodology may also be applicable to other antigen-antibody combinations, in immunoaffinity chromatography for selective purification of a protein or in an immunosensor for detection of a protein.     

Mimicking photosynthesis in a computationally designed synthetic metalloprotein

While advances in protein design have made possible the construction of protein architectures with nativelike properties and predictable structures and function, there are as of yet no examples of functional, protein-based, solar energy conversion systems. This communication describes the design and characterization of an artificial reaction center (RC) protein that closely resembles the function of the natural photosynthetic RC. The synthetic protein, designed by the protein design program CORE, participates in multiple reduction/oxidation cycles with exogenous acceptors/donors following photoexcitation. The designed metalloprotein, aRC, consists of a tetrahelical bundle functionalized with two bis-histidine bound metal cofactors: a Ru(bpy)2 moiety and a heme group. Two distinct bis-histidine binding sites were engineered for each of these metal centers. Photoexcitation of aRC results in rapid ET from the RuII complex to the heme group (kET >/= 5 x 1010 s-1) yielding a long-lived (70 ns) charge-separated state (CSS), RuIII/FeII. This long-lived CSS participates in subsequent ET reactions with exogenous donors and acceptors in multiple photocycles, thus mimicking the basic function of native photosynthetic RCs. This study illustrates the successful design and construction of a protein-based functional charge separation device using a combination of automated computational protein design and knowledge of the engineering principles of biological electron tunneling extracted from natural electron-transfer systems. To our knowledge, this represents the first example of a functional protein-based artificial reaction center.     

Functional tuning and expanding of myoglobin by rational protein design

Rational protein design is a powerful strategy, not only for revealing the structure and function relationship of natural metallo-proteins, but also for creating artificial metalloproteins with improved properties and functions. Myoglobin (Mb), a small heme protein created by nature with diverse functions, has been shown to be an ideal scaffold for rational protein design. The progress reviewed herein includes fine-tuning its native functions of O₂ binding and transport, peroxidase activity and nitrite reductase (NIR) activity, and rational expanding its functionalities to peroxygenase, heme-copper oxidase (HCO), nitric oxide reductase (NOR), as well as hydroxylamine reductase. These studies have enhanced our understanding of how metalloproteins work in nature, and provided insights for rational design of functional metalloproteins for practical applications in the future.     

Identification of Protein Functional Regions

Protein sequence stores the information relative to both functionality and stability, thus making it difficult to disentangle the two contributions. However, the identification of critical residues for function and stability has important implications for the mapping of the proteome interactions, as well as for many pharmaceutical applications, e. g. the identification of ligand binding regions for targeted pharmaceutical protein design. In this work, we propose a computational method to identify critical residues for protein functionality and stability and to further categorise them in strictly functional, structural and intermediate. We evaluate single site conservation and use Direct Coupling Analysis (DCA) to identify co-evolved residues both in natural and artificial evolution processes. We reproduce artificial evolution using protein design and base our approach on the hypothesis that artificial evolution in the absence of any functional constraint would exclusively lead to site conservation and co-evolution events of the structural type. Conversely, natural evolution intrinsically embeds both functional and structural information. By comparing the lists of conserved and co-evolved residues, outcomes of the analysis on natural and artificial evolution, we identify the functional residues without the need of any a priori knowledge of the biological role of the analysed protein.     

2-(二(2-氨乙基)氨基)乙醇Pd~Ⅱ配合物与小肽的相互作用

本文采用电喷雾质谱研究了带羟乙基侧臂二乙烯三胺Pde~Ⅱ配合物分别与含有硫原子侧链或咪唑侧链的二肽Met-Ala、乙酰化三肽AcGHG和AcGHL,十一肽Mp-11和三十肽氧化胰岛素B链的相互作用,发现该配合物能较好地结合这些多肽.但未能促使它们发生水解反应.比较相应铜配合物及二乙烯三胺铜配合物的切割性能,提示羟乙基侧臂单独难以实现相邻肽键的切割,高配位数金属中心对肽键羰基的活化与羟基的协同进攻是该类配合物切割多肽的可能机制.研究可为人工金属肽酶的发展提供新的设计战略.     

Structural and functional properties of designed globins

De novo design of artificial proteins is an essential approach to elucidate the principles of protein architecture and to understand specific functions of natural proteins and also to yield novel molecules for medical and industrial aims. We have designed artificial sequences of 153 amino acids to fit the main-chain framework of the sperm whale myoglobin structure based on the knowledge-based energy functions to evaluate the compatibility between protein tertiary structures and amino acid sequences. The synthesized artificial globins bind a single heme per protein molecule as designed, which show well-defined electrochemical and spectroscopic features characteristic of proteins with a low-spin heme. Redox and ligand binding reactions of the artificial heme proteins were investigated and these heme-related functions were found to vary with their structural uniqueness. Relationships between the structural and functional properties are discussed.     

Color tuning in photofunctional proteins

Depending on protein environment, a single photofunctional chromophore shows a wide variation of photoabsorption/emission energies. This photobiological phenomenon, known as color tuning, is observed in human visual cone pigments, firefly luciferase, and red fluorescent protein. We investigate the origin of color tuning by quantum chemical calculations on the excited states: symmetry-adapted cluster-configuration interaction (SAC-CI) method for excited states and a combined quantum mechanical (QM)/molecular mechanical (MM) method for protein environments. This Minireview summarizes our theoretical studies on the above three systems and explains a common feature of their color-tuning mechanisms. It also discuss the possibility of artificial color tuning toward a rational design of photoabsorption/emission properties.     

Process optimization and kinetics study for photocatalytic ciprofloxacin degradation using TiO2 nanoparticle: A comparative study of Artificial Neural Network and Surface Response Methodology

The photocatalytic degradation of ciprofloxacin was investigated by developing a predictive mathematical model using response surface methodology and an artificial neural network. The four independent variables involve solution pH, reaction time, catalyst dose, and initial antibiotic concentration considered as factors in central composite design to observe the response in the form of antibiotic degradation. Accordingly, at an optimum antibiotic concentration of 5.02 mg/L, catalyst dose of 44.51 mg/L, solution pH of 5.04, and reaction time of 75.80 min, the photocatalysis method achieved a ciprofloxacin degradation of 88.30%. The experimental outputs were very much consistent along with the predicted output of experiments through response surface methodology (R² = 0.9969) and artificial neural network (R² = 0.975). The adsorption isotherm and kinetic study reveal that Langmuir isotherm and pseudo-second-order kinetic models respectively were best fitted for degradation of ciprofloxacin through photocatalysis. The finding provides a novel method for evaluating the photocatalysis process for the optimization of ciprofloxacin antibiotic removal from pharmaceutical waste using experiments and computer simulation tools.     

Design of artificial metalloenzymes using non-covalent insertion of a metal complex into a protein scaffold

Construction of artificial metalloenzymes is one of the most attractive targets in the field of inorganic and catalytic chemistry, since they show remarkable chemoselectivity and reactivity in aqueous media. For the purpose, covalent modification of protein and cofactors have usually been utilized to attach a metal complex(es) to a protein scaffold. This article focuses on non-covalent insertion of metal complexes into protein environments. The discussion includes the screening of stable metal complex/protein composites, crystal structures, molecular design for regulating enantioselectivity of the target catalytic reactions. Our recent results show that the non-covalent conjugation will provide us a new way in semi-synthesis of artificial metalloenzymes.     

Synthesis and properties of biomimetic poly(L‐glutamate)‐b‐poly(2‐acryloyloxyethyllactoside)‐b‐poly(L‐glutamate) triblock copolymers

A novel class of biomimetic glycopolymer–polypeptide triblock copolymers [poly(L ‐glutamate)–poly(2‐acryloyloxyethyllactoside)–poly(L ‐glutamate)] was synthesized by the sequential atom transfer radical polymerization of a protected lactose‐based glycomonomer and the ring‐opening polymerization of β‐benzyl‐L ‐glutamate N‐carboxyanhydride. Gel permeation chromatography and nuclear magnetic resonance analyses demonstrated that triblock copolymers with defined architectures, controlled molecular weights, and low polydispersities were successfully obtained. Fourier transform infrared spectroscopy of the triblock copolymers revealed that the α‐helix/β‐sheet ratio increased with the poly(benzyl‐L ‐glutamate) block length. Furthermore, the water‐soluble triblock copolymers self‐assembled into lactose‐installed polymeric aggregates; this was investigated with the hydrophobic dye solubilization method and ultraviolet–visible analysis. Notably, this kind of aggregate may be useful as an artificial polyvalent ligand in the investigation of carbohydrate–protein recognition and for the design of site‐specific drug‐delivery systems. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 5754–5765, 2004     

Investigating the effect of hydrogen bonding environments in amide cleavage reactions at zinc(II) complexes with intramolecular amide oxygen co-ordination

Amide oxygen co-ordination to a zinc(II) ion around a hydrogen bonding microenvironment is a common structural/functional feature of metalloproteases. We report two strategies to position hydrogen bonding groups in the proximity of a zinc(II)-bound amide oxygen, and we investigate their effect on the stability of the amide group. Polydentate tripodal ligands (6-R1-2-pyridylmethyl)-R2 (R1= NHCOtBu, R2= N(CH2-py-6-X)2 X = H L1, X = NH2, H L2, X = NH2 L3) form [(L)Zn]2+ cations (L =L1, 1; L2, 2; L3, 3) with intramolecular amide oxygen co-ordination (1-3), and intramolecular N-H...O=C(amide) hydrogen bonding (2, 3) rigidly fixed by the ligand framework. 1-3 undergo cleavage of the tert-butyl amide upon addition of Me4NOH.5H2O (1 equiv.) in methanol at 50(1) degrees C. Under these conditions the half-life, t(1/2), of the amide bond is 0.4 h for 1, 9 h for 2 and 320 h for 3. Mononuclear zinc(II) complexes of (6-NHCOtBu-2-pyridylmethyl)-R2(R2= N(CH2CH2)2S) L4 and chelating N2 ligands without hydrogen bonding groups (1,10-phenanthroline L5, 2-(aminomethyl)pyridine L6) as control compounds, and with an amino hydrogen bonding group (6-amino-2-(aminomethyl)pyridine L7) have been synthesised. Amide cleavage is in this case faster at the zinc(II) complex with the amino hydrogen bonding group. Thus, hydrogen bonding environments can both accelerate and slow down amide bond cleavage reactions at zinc(II) sites. Importantly, the magnitude of the effect exerted by the hydrogen bonding environments was found to be significant; 800-fold rate difference. This result highlights the importance of hydrogen bonding environments around metal centres in amide cleavage reactions, which may be relevant to the chemistry of natural metalloproteases and applicable to the design of more efficient artificial protein cleaving agents.     

Self-Assembly of DNA–Peptide Supermolecules: Coiled-Coil Peptide Structures Templated by d-DNA and l-DNA Triplexes Exhibit Chirality-Independent but Orientation-Dependent Stabilizing Cooperativity

DNA nanostructures have been designed and used in many different applications. However, the use of nucleic acid scaffolds to promote the self-assembly of artificial protein mimics is only starting to emerge. Herein five coiled-coil peptide structures were templated by the hybridization of a d -DNA triplex or its mirror-image counterpart, an l -DNA triplex. The self-assembly of the desired trimeric structures in solution was confirmed by gel electrophoresis and small-angle X-ray scattering, and the stabilizing synergy between the two domains was found to be chirality-independent but orientation-dependent. This is the first example of using a nucleic acid scaffold of l -DNA to template the formation of artificial protein mimics. The results may advance the emerging POC-based nanotechnology field by adding two extra dimensions, that is, chirality and polarity, to provide innovative molecular tools for rational design and bottom-up construction of artificial protein mimics, programmable materials and responsive nanodevices.     

Self‐Assembly of Proteins: Towards Supramolecular Materials

The study of protein self‐assembly has attracted great interest over the decades, due to the important role that proteins play in life. In contrast to the major achievements that have been made in the fields of DNA origami, RNA, and synthetic peptides, methods for the design of self‐assembling proteins have progressed more slowly. This Concept article provides a brief overview of studies on native protein and artificial scaffold assemblies and highlights advances in designing self‐assembling proteins. The discussions are focused on design strategies for self‐assembling proteins, including protein fusion, chemical conjugation, supramolecular, and computational‐aided de novo design.