Decoration of discretely immobilized cowpea mosaic virus with luminescent quantum dots

This report describes two related methods for decorating cowpea mosaic virus (CPMV) with luminescent semiconductor nanocrystals (quantum dots, QDs). Variants of CPMV are immobilized on a substrate functionalized with NeutrAvidin using modifications of biotin-avidin binding chemistry in combination with metal affinity coordination. For example, using CPMV mutants expressing available 6-histidine sequences inserted at loops on the viral coat protein, we show that these virus particles can be specifically immobilized on NeutrAvidin functionalized substrates in a controlled fashion via metal-affinity coordination. To accomplish this, a hetero-bifunctional biotin-NTA moiety, activated with nickel, is used as the linker for surface immobilization of CPMV (bridging the CPMVs' histidines to the NeutrAvidin). Two linking chemistries are then employed to achieve CPMV decoration with hydrophilic CdSe-ZnS core-shell QDs; they target the histidine or lysine residues on the exterior virus surface and utilize biotin-avidin interactions. In the first scheme, QDs are immobilized on the surface-tethered CPMV via electrostatic attachment to avidin previously bound to the virus particle. In the second strategy, the lysine residues common to each viral surface asymmetric unit are chemically functionalized with biotin groups and the biotinylated CPMV is discretely immobilized onto the substrate via NeutrAvidin-biotin interactions. The biotin units on the upper exposed surface of the immobilized CPMV then serve as capture sites for QDs conjugated with a mixture of avidin and a second protein, maltose binding protein, which is also used for QD-protein conjugate purification. Characterization of the assembled CPMV and QD structures is presented, and the potential uses for protein-coated QDs functionalized onto this symmetrical virion nanoscaffold are discussed.     

Natural supramolecular building blocks. Wild-type cowpea mosaic virus

Cowpea mosaic virus (CPMV) can be isolated in gram quantities, possesses a structure that is known to atomic resolution, and is quite stable. It is therefore of potential use as a molecular entity in synthesis, particularly as a building block on the nanochemical scale. CPMV was found to possess a lysine residue with enhanced reactivity in each asymmetric unit, and thus 60 such lysines per virus particle. The identity of this residue was established by a combination of acylation, protein digestion, and mass spectrometry. Under forcing conditions, up to four lysine residues per asymmetric unit can be addressed. In combination with engineered cysteine reactivity described in the accompanying paper, this provides a powerful platform for the alteration of the chemical and physical properties of CPMV particles.     

New addresses on an addressable virus nanoblock; uniquely reactive Lys residues on cowpea mosaic virus

Cowpea mosaic virus (CPMV) is a robust, icosahedrally symmetric platform successfully used for attaching a variety of molecular substrates including proteins, fluorescent labels, and metals. The symmetric distribution and high local concentration of the attached molecules generates novel properties for the 30 nm particles. We report new CPMV reagent particles generated by systematic replacement of surface lysines with arginine residues. The relative reactivity of each lysine on the native particle was determined, and the two most reactive lysine residues were then created as single attachment sites by replacing all other lysines with arginine residues. Structural analysis of gold derivatization not only corroborated the specific reactivity of these unique lysine residues but also demonstrated their dramatically different presentation environment. Combined with site-directed cystine mutations, it is now possible to uniquely double label CPMV, expanding its use as an addressable nanoblock.     

Separation and recovery of intact gold-virus complex by agarose electrophoresis and electroelution: application to the purification of cowpea mosaic virus and colloidal gold complex

Colloidal gold has been coupled to a mutant cowpea mosaic virus (CPMV), which contains 60 cysteine residues on the surface. A purification process was developed to separate the gold-containing viral nanoblocks (VNBs) from the free gold. Agarose electrophoresis was utilized to separate the mixture followed by electroelution of the desired sample to recover the intact virus. Mobility of Au-VNB and free colloidal gold was facilitated by the addition of thioctic acid (TA). 30% of the gold-containing virus was recovered after electroelution as determined by absorbance measurements. Histogram analysis of transmission electron microscopy (TEM) images demonstrated the efficient separation of gold-containing virus from free gold. TEM and scanning electron microscopy (SEM) images indicated that the virus was recovered intact. Monodisperse spherical particles of nominal size of 45 nm were observed under SEM.     

On-line liquid chromatography/electrospray tandem mass spectrometry to investigate acrylamide adducts with cysteine residues: implications for polyacrylamide gel electrophoresis separations of proteins

Adduction between acrylamide and cysteine residues is a post-translational modification associated with proteins separated by gel electrophoresis. In the present article, three model peptides containing 2–4 cysteine residues were reduced with dithiothreitol, incubated with acrylamide monomers and examined by on-line liquid chromatography coupled to electrospray tandem mass spectrometry. Each of the solutions examined in this work revealed the presence of four distinct components: the free peptide, two different peptide–acrylamide 1:1 adducts involving two cysteine residues at different positions within the same sequence, and peptide–acrylamide 1:2 adducts. The use of liquid chromatography allowed the separation of components which differed only by the site of complexation of acrylamide, while the application of tandem mass spectrometry furnished reliable sequencing information permitting the identification of most cysteine residues involved in such complexation. © 1998 John Wiley & Sons, Ltd.     

Cowpea mosaic virus capsid: a promising carrier for the development of carbohydrate based antitumor vaccines

Immunotherapy targeting tumor cell surface carbohydrates is a promising approach for cancer treatment. However, the low immunogenecity of carbohydrates presents a formidable challenge. We describe here the enhancement of carbohydrate immunogenicity by an ordered display on the surface of the cowpea mosaic virus (CPMV) capsid. The Tn glycan, which is overexpressed on numerous cancer cell surfaces, was selected as the model antigen for our study. Previously it has been shown that it is difficult to induce a strong T cell-dependent immune response against the monomeric form of Tn presented in several ways on different carriers. In this study, we first synthesized Tn antigens derivatized with either a maleimide or a bromoacetamide moiety that was conjugated selectively to a cysteine mutant of CPMV. The glycoconjugate was then injected into mice and pre- and post-immune antibody levels in the mice sera were measured by enzyme-linked immunosorbant assays. High total antibody titers and, more importantly, high IgG titers specific for Tn were obtained in the post-immune day 35 serum, suggesting the induction of T cell-dependent antibody isotype switching by the glycoconjugate. The antibodies generated were able to recognize Tn antigens presented in their native conformations on the surfaces of both MCF-7 breast cancer cells and the multidrug resistant breast cancer cell line NCI-ADR RES. These results suggest that the CPMV capsid can greatly enhance the immunogenicity of weak antigens such as Tn and this can provide a promising tool for the development of carbohydrate based anti-cancer vaccines.     

Photocontrol of the collagen triple helix: synthesis and conformational characterization of bis-cysteinyl collagenous peptides with an azobenzene clamp

For the photomodulation of the collagen triple helix with an azobenzene clamp, we investigated various collagenous peptides consisting of ideal (Gly-Pro-Hyp) repeats and containing cysteine residues in various positions for a side chain-to-side chain crosslink with a suitable chromophore derivative. Comparative conformational analysis of these cysteine peptides indicated an undecarepeat peptide with two cysteine residues located in the central portion in i and i+7 positions and flanked by (Gly-Pro-Hyp) repeat sequences as the most promising for the cross-bridging experiments. In aqueous alcoholic solution the azobenzene-undecarepeat peptide formed a stable triple helix in equilibrium with the monomeric species as a trans-azobenzene isomer, whereas photoisomerization to the cis isomer leads to unfolding of at least part of the triple helix. Furthermore, the residual supercoiled structure acts like an intermolecular knot, thus making refolding upon cis-to-trans isomerization a concentration-independent fast event. Consequently, these photoswitchable collagenous systems should be well suited for time-resolved studies of folding/unfolding of the collagen triple helix under variable thermodynamic equilibria.     

Rapid chemoselective bioconjugation through oxidative coupling of anilines and aminophenols

A highly efficient protein bioconjugation method is described involving addition of anilines to o-aminophenols in the presence of sodium periodate. The reaction takes place in aqueous buffer at pH 6.5 and can reach high conversion in 2-5 min. The major product was characterized using X-ray crystallography, which revealed that an unprecedented oxidative ring contraction occurs after the coupling step. The compatibility of the reaction with protein substrates has been demonstrated through attachment of small molecules, polymer chains, and peptides to p-aminophenylalanine residues introduced into viral capsids through amber stop codon suppression. Coupling of anilines to o-aminophenol groups derived from tyrosine residues is also described. The compatibility of this method with thiol modification chemistry is shown through attachment of a near-IR fluorescent chromophore to cysteine residues inside the viral capsid shells, followed by attachment of integrin-targeting RGD peptides to anilines on the exterior surface.     

Selective thioether macrocyclization of peptides having the N-terminal 2-chloroacetyl group and competing two or three cysteine residues in translation

The mode of thioether macrocyclization of peptides containing an N-terminal 2-chloroacetyl group and two or three competing cysteine residues at downstream positions has been extensively studied, leading to a strategy for designated formation of overlapping-bicyclic peptides or dumbbell-type bicyclic peptides.     

Purification and initial characterization of a novel protein with factor Xa activity from Lonomia obliqua caterpillar spicules

A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined.     

Peptide modification through site-selective residue interconversion: application of the rhodium-catalysed 1,4-addition of aryl siloxanes and boronates

The site-selective interconversion of serine and cysteine residues of di- and tripeptides into phenylalanine derivatives, bearing a range of functionalities, has been achieved in high yield and selectivity through the common dehydroalanine intermediate. Through the application and development of the rhodium-catalysed 1,4-addition to α,β-dehydroamino acid moieties with organometallic nucleophiles, a variety of peptides have been successfully modified to contain unnatural amino acid residues in pre-designated residue positions.     

Folic acid-mediated targeting of cowpea mosaic virus particles to tumor cells

Cowpea mosaic virus (CPMV) is a well-characterized nanoparticle that has been used for a variety of nanobiotechnology applications. CPMV interacts with several mammalian cell lines and tissues in vivo. To overcome natural CPMV targeting and redirect CPMV particles to cells of interest, we attached a folic acid-PEG conjugate by using the copper-catalyzed azide-alkyne cycloaddition reaction. PEGylation of CPMV completely eliminated background binding of the virus to tumor cells. The PEG-folate moiety allowed CPMV-specific recognition of tumor cells bearing the folate receptor. In addition, by testing CPMV formulations with different amounts of the PEG-FA moiety displayed on the surface, we show that higher-density loading of targeting ligands on CPMV may not be necessary for efficient targeting to tumor cells. These studies help to define the requirements for efficiently targeting nanoparticles and protein cages to tumors.     

Loss of selenium from selenoproteins: conversion of selenocysteine to dehydroalanine in vitro

Characterization of reduced and alkylated rat selenoprotein P by mass spectrometry yielded selenopeptides from which one or more selenium atoms were missing. Predicted selenopeptide mass peaks were accompanied by peaks corresponding to the conversion of one or more selenocysteine residues to dehydroalanine(s). Experiments were carried out to determine whether this loss of selenium occurred in vitro. A selenopeptide was isolated that contained two selenocysteine residues that were both in selenide-sulfide linkages with cysteine residues. After the peptide had been reduced and alkylated, in addition to the predicted mass peak with both selenocysteine residues present, two mass peaks were detected at positions expected for conversion of one and two selenocysteine residues of this selenopeptide to dehydroalanine residues, which was confirmed by tandem mass spectrometry. Similar findings were obtained from a study of another selenoprotein, rat plasma glutathione peroxidase. These results indicate that selenium atoms are lost from selenoproteins during purification and characterization. The loss of selenium from selenoproteins is probably through the mechanism of oxidation of selenocysteine residue to selenoxide followed by syn-beta-elimination of selenenic acid during sample processing.     

采用电活性标记物N-(2-乙基-二茂铁)马来酰亚胺检测与表面固定的 一致性双链DNA特异性作用的p53蛋白质

建立了电化学检测表面固定捕获的野生型p53蛋白质的方法. 首先在金电极表面形成巯基化的单链DNA探针/己硫醇(HT)混合自组装膜, 随后巯基化的单链DNA探针与溶液中序列匹配的靶点DNA杂交, 所形成的一致性双链DNA捕获溶液中的野生型p53蛋白质. p53分子表面的半胱氨酸残基采用巯基特异性试剂N-(2-乙基-二茂铁)马来酰亚胺(Fc-Mi)进行衍生. 通过检测二茂铁的电化学信号来指示p53与一致性双链DNA之间的特异性相互作用. p53蛋白质与双链DNA的键合程度取决于双链DNA的序列. 该方法可检测的p53最低浓度为1.33 nmol•L－1.     

hCB2 ligand-interaction landscape: cysteine residues critical to biarylpyrazole antagonist binding motif and receptor modulation

The human cannabinoid 2 GPCR (hCB2) is a prime therapeutic target. To define potential cysteine-related binding motifs critical to hCB2-ligand interaction, a library of hCB2 cysteine-substitution mutants and a novel, high-affinity biarylpyrazole hCB2 antagonist/inverse agonist (AM1336) functionalized to serve as a covalent affinity probe to target cysteine residues within (or in the microenvironment of) its hCB2 binding pocket were generated. The data provide direct experimental demonstration that both hCB2 TMH7 cysteines [i.e., C7.38(284) and C7.42(288)] are critical to optimal hCB2-AM1336 binding interaction and AM1336 pharmacological activity in a cell-based functional assay (cAMP formation). Elongating the AM1336 aliphatic side chain generated another novel?hCB2 inverse agonist that binds covalently and selectively to C7.42(288) only. Identification of specific cysteine residues critical to hCB2 ligand interaction and function informs the structure-based design of hCB2-targeted medicines.     

Light-initiated hydroxylation of lauric acid using hybrid P450 BM3 enzymes

We have developed hybrid P450 BM3 enzymes consisting of a Ru(II)-diimine photosensitizer covalently attached to non-native single cysteine residues of P450 BM3 heme domain mutants. These enzymes are capable, upon light activation, of selectively hydroxylating lauric acid with 40 times higher total turnover numbers compared to the peroxide shunt.     

Locating Pb2+ and Zn2+ in Zinc Finger-Like Peptides Using Mass Spectrometry

The binding preferences of Pb²⁺and Zn²⁺ in doubly charged complexes with zinc finger-like 12-residue peptides (Pep), [M_n(Pep-2(n-1)H)]²⁺ have been explored using tandem mass spectrometry. The peptides were synthesized strategically by blocking the N-terminus with an acetyl group and with four cysteine and/or histidine residues in positions 2, 5, 8, and 11, arranged in different motifs: CCHH, CHCH, and CCCC. The MS² spectra of the Pb²⁺ and Zn²⁺ complexes show multiple losses of water and a single methane loss and these provide a sensitive method for locating the metal dication and so elucidating its coordination. The elimination of a methane molecule indicated the position of the metal at the Cys2 residue. Whereas lead was observed to preferentially bind to cysteine residues, zinc was found to primarily bind to histidine residues and secondarily to cysteine residues. Preferential binding of lead to cysteine is preserved in the complexes with more than one Pb²⁺. Key to the mechanism of the loss of water and methane is the metal dication withdrawing electrons from the proximal amidic nitrogen. This acidic nitrogen loses its hydrogen to an amidic oxygen situated four atoms away leading to formation of a five-member ring and the elimination of water.      

Dielectrophoretic investigation of plant virus particles: Cow Pea Mosaic Virus and Tobacco Mosaic Virus

This paper reports experimental results on the dielectrophoretic (DEP) behaviour on two nonenveloped plant viruses of different geometrical shapes, namely Cow Pea Mosaic Virus (CPMV) and Tobacco Mosaic Virus (TMV). The DEP properties of carboxy-modified latex beads of the same size are also reported. The DEP properties of single particles were obtained from measurement of the frequency at which the DEP force on a particle goes to zero (the crossover frequency). The DEP behaviour of particle ensembles was also measured using image processing. The dielectric properties of the particles were evaluated from the DEP data. The surface conductance was found to be 0.3 nS for CPMV, 0.38 nS for TMV, and 0.52 nS for 27 nm diameter carboxy-latex beads. Data analysis has shown that the optimal condition for separation of TMV and CPMV is a low-conductivity suspending medium - below 1 mS/m.     

IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis

The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity, flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 microM, respectively. EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S](+) cluster in IspH protein. Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000.     

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.