Flavin Adenine Dinucleotide and N5,N10‐Methenyltetrahydrofolate are the in planta Cofactors of Arabidopsis thaliana Cryptochrome 3

Members of the cryptochrome/photolyase family (CPF) of proteins utilize noncovalently bound light‐absorbing cofactors for their biological function. Usually, the identity of these cofactors is determined after expression in heterologous systems leaving the question unanswered whether these cofactors are identical to the indigenous ones. Here, cryptochrome 3 from Arabidopsis thaliana was expressed as a fusion with the green fluorescent protein in Arabidopsis plants. Besides the confirmation of the earlier report of its localization in chloroplasts, our data indicate that fractions of the fusion protein are present in the stroma and associated with thylakoids, respectively. Furthermore, it is shown that the fusion protein expressed in planta contains the same cofactors as the His₆‐tagged protein expressed in Escherichia coli, that is, flavin adenine dinucleotide and N⁵,N¹⁰‐methenyltetrahydrofolate. This demonstrates that the heterologously expressed cryptochrome 3, characterized in a number of previous studies, is a valid surrogate of the corresponding protein expressed in plants. To our knowledge, this is also a first conclusive analysis of cofactors bound to an Arabidopsis protein belonging to the CPF and purified from plant tissue.     

Conformational control of cofactors in nature--functional tetrapyrrole conformations in the photosynthetic reaction centers of purple bacteria

Chemically identical tetrapyrrole cofactors such as hemes and chlorophylls participate in functionally diverse biological roles. An analysis of the available protein structural data for the bacteriochlorophylls in the photosynthetic reaction center gives statistically reliable evidence of the hypothesis that the protein induced cofactor conformation is a modulator of the bio-molecular function of each reaction center. The results serve as a general model to illustrate conformational control of tetrapyrrole cofactors in other proteins.     

Chemical Imaging of RNA-Tau Amyloid Fibrils at the Nanoscale Using Tip-Enhanced Raman Spectroscopy

In the presence of cofactors, tau protein can form amyloid deposits in the brain which are implicated in many neurodegenerative disorders. Heparin, lipids, and RNA are used to recreate tau aggregates in vitro from recombinant protein. However, the mechanism of interaction of these cofactors and the interactions between cofactors and tau are poorly understood. Herein, we use tip-enhanced Raman spectroscopy (TERS) to visualize the spatial distribution of adenine, protein secondary structure, and amino acids (arginine, lysine and histidine) in single polyadenosine (polyA)-induced tau fibrils with nanoscale spatial resolution (<10–20 nm). Based on reference unenhanced and surface-enhanced Raman spectra, we show that the polyA anionic cofactor is incorporated in the fibril structure and seems to be superficial to the β-sheet core, but nonetheless enveloped within the random-coiled fuzzy coat. TERS images also prove the colocalization of positively charged arginine, lysine, and histidine amino acids and negatively charged polyA, which constitutes an important step forward to better comprehend the action of RNA cofactors in the mechanism of formation of toxic tau fibrils. TERS appears as a powerful technique for the identification of cofactors in individual tau fibrils and their mode of interaction.     

Combined use of trypsin-agarose affinity chromatography and reversed-phase high-performance liquid chromatography for the purification of single-chain protease inhibitor from corn seeds

We have developed a large-scale method for recovering the corn inhibitor of trypsin and activated Hageman factor from a trypsin-agarose column predominantly in the single-chain form. To do so, inhibitor retained by the column was eluted with 1.0 M glycine buffer, pH 2.1. We have used reversed-phase high-performance liquid chromatography to further purify the inhibitor eluted from the trypsin-agarose column by separating the single-chain inhibitor from two-chain inhibitor (a small amount of which is present in the preparation after trypsin-agarose chromatography) and from still smaller amounts of another protein (apparently trypsin) that appears as a contaminant during trypsin-agarose chromatography.     

Biosynthetic Approaches towards the Design of Artificial Hydrogen-Evolution Catalysts

Hydrogen is a clean and sustainable form of fuel that can minimize our heavy dependence on fossil fuels as the primary energy source. The need of finding greener ways to generate H₂ gas has ignited interest in the research community to synthesize catalysts that can produce H₂ by the reduction of H⁺. The natural H₂ producing enzymes hydrogenases have served as an inspiration to produce catalytic metal centers akin to these native enzymes. In this article we describe recent advances in the design of a unique class of artificial hydrogen evolving catalysts that combine the features of the active site metal(s) surrounded by a polypeptide component. The examples of these biosynthetic catalysts discussed here include i) assemblies of synthetic cofactors with native proteins; ii) peptide-appended synthetic complexes; iii) substitution of native cofactors with non-native cofactors; iv) metal substitution from rubredoxin; and v) a reengineered Cu storage protein into a Ni binding protein. Aspects of key design considerations in the construction of these artificial biocatalysts and insights gained into their chemical reactivity are discussed.     

Proteomic Studies of Syk-Interacting Proteins Using a Novel Amine-Specific Isotope Tag and GFP Nanotrap

Green fluorescent protein (GFP) and variants have become powerful tools to study protein localization, interactions, and dynamics. We present here a mass spectrometry-based proteomics strategy to examine protein–protein interactions using anti-GFP single-chain antibody V_HH in a combination with a novel stable isotopic labeling reagent, isotope tag on amino groups (iTAG). We demonstrate that the single-chain V_HH (GFP nanotrap) allows us to identify interacting partners of the Syk protein-tyrosine kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified include CrkL, BLNK, α- and β-tubulin, Csk, RanBP5 and DJ-1. The iTAG reagents were prepared with simple procedures and characterized with high accuracy in the determination of peptides in model peptide mixtures and as well as in complex mixture. Applications of the iTAG method and GFP nanotrap to an analysis of the nucleocytoplasmic trafficking of Syk led to the identification of location-specific associations between Syk and multiple proteins. While the results reveal that the new quantitative proteomic strategy is generally applicable to integrate protein interaction data with subcellular localization, extra caution should be taken in evaluating the results obtained by such affinity purification strategies as many interactions appear to occur following cell lysis.     

Role of cofactors in folding of the blue-copper protein azurin

Many proteins in living cells coordinate cofactors, such as metal ions, to attain their activity. Since the cofactors in such cases often can interact with their corresponding unfolded polypeptides in vitro, it is important to unravel how cofactors modulate protein folding. In this review, I will discuss the role of cofactors in folding of the blue-copper protein Pseudomonas aeruginosa azurin. In the case of both copper (Cu(II) and Cu(I)) and zinc (Zn(II)), the metal can bind to unfolded azurin. The residues involved in copper (Cu(II) and Cu(I)) coordination in the unfolded state have been identified as Cys112, His117, and Met121. The affinities of Cu(II), Cu(I), and Zn(II) are all higher for the folded than for the unfolded azurin polypeptide, resulting in metal stabilization of the native state as compared to the stability of apo-azurin. Cu(II), Zn(II), and several apo forms of azurin all fold in two-state kinetic reactions with roughly identical polypeptide-folding speeds. This suggests that the native-state beta-barrel topology, not cofactor interactions or thermodynamic stability, determines azurin's folding barrier. Nonetheless, copper binds much more rapidly (i.e., 4 orders of magnitude) to unfolded azurin than to folded azurin. Therefore, the fastest route to functional azurin is through copper binding before polypeptide folding; this sequence of events may be the relevant biological pathway.     

Prediction of reorganization free energies for biological electron transfer: a comparative study of Ru-modified cytochromes and a 4-helix bundle protein

The acceleration of electron transfer (ET) rates in redox proteins relative to aqueous solutes can be attributed to the protein's ability to reduce the nuclear response or reorganization upon ET, while maintaining sufficiently high electronic coupling. Quantitative predictions of reorganization free energy remain a challenge, both experimentally and computationally. Using density functional calculations and molecular dynamics simulation with an electronically polarizable force field, we report reorganization free energies for intraprotein ET in four heme-containing ET proteins that differ in their protein fold, hydrophilicity, and solvent accessibility of the electron-accepting group. The reorganization free energies for ET from the heme cofactors of cytochrome c and b(5) to solvent exposed Ru-complexes docked to histidine residues at the surface of these proteins fall within a narrow range of 1.2-1.3 eV. Reorganization free energy is significantly lowered in a designed 4-helix bundle protein where both redox active cofactors are protected from the solvent. For all ET reactions investigated, the major components of reorganization are the solvent and the protein, with the solvent contributing close to or more than 50% of the total. In three out of four proteins, the protein reorganization free energy can be viewed as a collective effect including many residues, each of which contributing a small fraction. These results have important implications for the design of artificial electron transport proteins. They suggest that reorganization free energy may in general not be effectively controlled by single point mutations, but to a large extent by the degree of solvent exposure of the ionizable cofactors.     

Single-chain single crystals

Single-chain single crystals of isotactic polystyrene and poly(ethylene oxide) were studied by using transmission electron microscopy, high resolution electron microscopy, electron diffraction. Single-chain single crystals were prepared by spreading a dilute solution of polymers on a water surface and collecting the resulting single-chain particles on copper grids, followed by isothermal crystallization. A statistical analysis of the sizes of single-chain crystals was found to match with the known molecular weight distribution of original sample, indicating the particles to be composed of single chain. Observation of the morphology and electron diffraction gave evidence of the single crystal nature. Regular-shaped single-chain crystals were obtained after isothermal crystallization for a longer time. By close observation, several types of morphologies were found for single-chain crystals of isotactic polystyrene and poly(ethylene oxide); in addition to the conventional morphologies observed for multi-chain crystals, new morphologies were observed in both cases. The morphologies of poly(ethylene oxide) were explained according to the crystal structure and twin modes. Tent-like single-chain crystals were often observed. Because of the small size of the crystals, they can avoid collapse on the substrate. The crystalline c-axis of single-chain crystals were found to orient preferably in the direction normal to the substrate. The investigation of electron diffraction and high resolution electron microscopy revealed that the structure of the single-chain crystals of isotactic polystyrene is the same as for multi-chain crystals. A reasonable explanation is given for the unusual resistance to electron irradiation and the missing of lower-index reflections. Regular periodic stripes were found on the top surface of single-chain crystal of isotactic polystyrene with an average periodic length in accordance with (220) spacing. In addition, a statistical thermodynamics theory was developed for single-chain crystal. It is found that the equilibrium dimensions are related to molecular weight and annealing temperature, while the equilibrium melting temperature depends on molecular weight.     

Nativelike structure in designed four alpha-helix bundles driven by buried polar interactions

We show that a single internal polar interaction per helix is sufficient to engender structural specificity in that helix in helical bundle proteins. Furthermore, we use histidine-binding cofactors of different shapes which bind directly into the core, demonstrating that this structural specificity is not the result of a prescribed complimentary, "knobs in holes" core packing. We show that we can switch structural specificity of individual helices on and off by ligating cofactors, singly and in pairs, which bind either one or two histidine ligands. To our knowledge, this is the first demonstration of such extensive manipulation of protein structure by ligand binding, an important result of general interest to those working with self-assembled molecular systems. Finally, as these proteins were designed without the use of computational modeling, we not only demonstrate that designing a uniquely structured cofactor binding protein is not as difficult as is generally believed, we have determined why this is so: hydrophobic core complementarity, which is very difficult to design, is not necessary. Instead, a much simpler design process entails the creation of core polar interactions which themselves can drive conformational specificity.     

Construction, Expression and Characterization of a Chimeric Protein Targeting Carcinoembryonic Antigen in Lung Cancer

The carcinoembryonic antigen(CEA) is an oncofetal glycoprotein known as an important clinical tumor marker and is overexpressed in several types of tumors, including colorectal and lung carcinomas. We constructed a chimeric protein that exhibits both specific binding and immune stimulating activities, by fusing staphylococcal enterotoxin A(SEA) to the C-terminus of an anti-CEA single-chain disulfide-stabilized Fv(scdsFv) antibody (single-chain-C-terminus/SEA, SC-C/SEA). The SC-C/SEA protein was expressed in...     

Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification

We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine–HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.     

Protein-cation interactions: structural and thermodynamic aspects

Cations are specifically recognized by numerous proteins. Cations may play a structural role, as cofactors stabilizing their binding partners, or a functional role, as cofactors activating their binding partners or being themselves involved in enzymatic reactions. Despite their small size, their charge density and their specific interaction with highly charged residues allow them to induce significant conformational changes on their binding proteins. The protein conformational change induced by cation binding may be as large as to account for the complete folding of a protein (as evidenced in Hepatitis C NS3 protease, or human rhinovirus 2A protease), and they may also trigger oligomerization (as in calcium-binding protein 1). Especially intriguing is the ability of cation-binding proteins of discriminating between very similar cations. In particular, calcium and magnesium are recognized by proteins with markedly different binding affinities and cause significantly different conformational changes and stabilization effects in the binding proteins (as in the fifth ligand binding repeat of the LDL receptor binding domain, calcium-binding protein 1, or parvalbumin). This article summarizes recent findings on the structural and energetic impact of cation binding to different proteins. A general framework can be envisaged in which cations can be considered as a special type of allosteric effectors able to modulate the functional properties of proteins, in particular the ability to interact with biological targets, by altering their conformational equilibrium.     

Force field development for cofactors in the photosystem II

We present a set of force field (FF) parameters compatible with the AMBER03 FF to describe five cofactors in photosystem II (PSII) of oxygenic photosynthetic organisms: plastoquinone‐9 (three redox forms), chlorophyll‐a, pheophytin‐a, heme‐b, and β‐carotene. The development of a reliable FF for these cofactors is an essential step for performing molecular dynamics simulations of PSII. Such simulations are important for the calculation of absorption spectrum and the further investigation of the electron and energy transfer processes. We have derived parameters for partial charges, bonds, angles, and dihedral‐angles from solid theoretical models using systematic quantum mechanics (QM) calculations. We have shown that the developed FF parameters are in good agreement with both ab initio QM and experimental structural data in small molecule crystals as well as protein complexes. © 2012 Wiley Periodicals, Inc.     

Cofactor-free detection of phosphatidylserine with cyclic peptides mimicking lactadherin

Cyclic peptides (cLacs) are designed to mimic the natural phosphatidylserine (PS) binding protein lactadherin. Unlike annexin V or its small molecule mimics, the cLac peptides selectively target PS-presenting membranes with no need for metal cofactors. We further show that a fluorophore-labeled cLac effectively stains early apoptotic cells. The small size and facile conjugation with a variety of imaging tracers make the cLac design promising for imaging cell death in vitro as well as in living organisms.     

Synthesis and preliminary investigations of the siRNA delivery potential of novel, single-chain rigid cationic carotenoid lipids

The success of nucleic acid delivery requires the development of safe and efficient delivery vectors that overcome cellular barriers for effective transport. Herein we describe the synthesis of a series of novel, single-chain rigid cationic carotenoid lipids and a study of their preliminary in vitro siRNA delivery effectiveness and cellular toxicity. The efficiency of siRNA delivery by the single-chain lipid series was compared with that of known cationic lipid vectors, 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (EPC) as positive controls. All cationic lipids (controls and single-chain lipids) were co-formulated into liposomes with the neutral co-lipid, 1,2-dioleolyl-sn-glycerol-3-phosphoethanolamine (DOPE). Cationic lipid-siRNA complexes of varying (+/-) molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/-) molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid represents a new class of transfection vector with excellent cell tolerance, accompanied with encouraging siRNA delivery efficiency.     

Intein-mediated purification of a recombinantly expressed peptide

A 26 amino acid peptide has successfully been purified via recombinant expression as an intein fusion protein accompanied by cleavage without the need for any exogenous proteases or cofactors, thus offering a practical, inexpensive approach to produce isotopically labelled peptides.