Heterologous expression and characterization of recombinant phytochrome from the green alga Mougeotia scalaris

The full-length apoprotein (124 kDa) and the chromophore-binding N-terminal half (66 kDa) of the phytochrome of the unicellular green alga Mougeotia scalaris have been heterologously expressed in the methylotrophic yeast Pichia pastoris. Assembly with the tetrapyrrole phycocyanobilin (PCB) yielded absorption maxima (for the full-length protein) at 646 and 720 nm for red- and far-red absorbing forms of phytochrome (Pr and Pfr), respectively, whereas the maxima of the N-terminal 66 kDa domain are slightly blueshifted (639 and 714 nm, Pr and Pfr, respectively). Comparison with an action spectrum reported earlier gives evidence that in Mougeotia, as formerly reported for the green alga Mesotaenium caldariorum, PCB constitutes the genuine chromophore. The full-length protein, when converted into its Pfr form and kept in the dark, reverted rapidly into the Pr form (lifetimes of 1 and 24 min, ambient temperature), whereas the truncated chromopeptide (66 kDa construct) was more stable and converted into Pr with time constants of 18 and 250 min. Also, time-resolved analysis of the light-induced Pfr formation revealed clear differences between both recombinant chromoproteins in the various steps involved. The full-length phytochrome showed slower kinetics in the long milliseconds-to-seconds time domain (with dominant Pfr formation processes of ca 130 and 800 ms), whereas for the truncated phytochrome the major component of Pfr formation had a lifetime of 32 ms.     

Control of the stability of Hydrogenobacter thermophilus cytochrome C(552) through alteration of the basicity of the N-terminal amino group of the polypeptide chain

In the denatured state of Hydrogenobacter thermophilus cytochrome c(552) (HT), the N-terminal amino group of the polypeptide chain is coordinated to the heme Fe in place of the axial Met, the His-N(term) form being formed [Tai, H., Munegumi, T., Yamamoto, Y. Inorg. Chem. 2009, 48, 331-338]. Since the His-N(term) form can be considered as an ordered residual structure in the denatured protein, its stability significantly influences the energy of the denatured state. In this study, the His-N(term) forms of the wild-type HT and its mutants possessing a series of amino acid residues at the N-terminal, such as N1D, N1E, and N1G, have been characterized to elucidate the physicochemical properties of the N-terminal residue responsible for the control of the thermodynamic stability of the His-N(term) form. The study revealed that the thermodynamic stability of the His-N(term) form depends highly on the basicity of the N-terminal amino group of the polypeptide chain in such a manner that an increase in the pK(a) value of the N-terminal amino group by 1 unit results in stabilization of the bond between heme Fe and the N-terminal amino group (Fe-N(term) bond) in the His-N(term) form by ～4 kJ mol(-1). The empirical hard and soft acid and base principle could account for the observed relationship between the pK(a) value of the N-terminal amino group and the stability of the Fe-N(term) bond in the His-N(term) form. In addition, the study demonstrated that the overall stability of the protein can be manipulated through the energy of the denatured protein by changing the thermodynamic stability of the His-N(term) form. Consequently, the overall stability of the protein has been shown to be controlled through alteration of the basicity of the N-terminal amino group of the polypeptide chain. These findings provide new insights into the stabilizing interactions in the denatured protein, which are relevant as to characterization of the protein stability and folding.     

Recombinant Glutamine Synthetase (GS) from <Emphasis Type="Italic">C. glutamicum</Emphasis> Existed as Both Hexamers &; Dedocamers and C-terminal His-tag Enhanced Inclusion Bodies Formation in <Emphasis Type="Italic">E. coli</Emphasis>

In order to investigate the effect of his-tag on glutamine synthetase (GS, EC 6.3.1.2) from Corynebacterium glutamicum, recombinant Escherichia coli strains overexpressing GSIM, HGSIM (GS fused with N-terminal his-tag), GSIMH (GS fused with C-terminal his-tag), and HGSIMH (GS fused with N-terminal & C-terminal his-tags) were constructed, respectively. Under similar expression conditions, GSIM and HGSIM were partially solubly expressed; no soluble GSIMH and HGSIMH were observed, based on the result of SDS-PAGE. Gel filtration of purified soluble HGSIM showed that hexamers and dedocamers coexisted in the quaternary structure of GS from C. glutamicum. Combined this result with the analysis of two GS crystal structure models, we hypothesized that C-terminal residues participated in GS folding after translation on ribosome. After the folding process, C-terminal residues were released again and exposed to solvent. Fused C-terminal his-tag interrupted the GS to fold into its correct conformation so that inclusion bodies formed.     

Generation, translocation, and action of nitric oxide in living systems

Nitric oxide (NO) is a gaseous diatomic radical that is involved in a wide range of physiological and pathological functions in biology. Conceptually, the biochemistry of NO can be separated into three stages: generation (stage 1), translocation (stage 2), and action (stage 3). In stage 1 the oxygenase domain of NO synthase converts L-arginine to L-citrulline and NO (g). Owing to its short-lived nature, this molecule is converted into a different nitrogen oxide such as NO(2), an organonitrosyl such as a nitrosothiol, or a metal nitrosyl such as a heme-nitrosyl, for transportation in stage 2. Each of these derivatives features unique physical characteristics, chemical reactivity, and biological activity. Upon delivery in stage 3, NO exerts its physiological or pathological function by reaction with biomolecules containing redox-active metals or other residues.     

Density Functional Theory Study of Water Diffusion and Clustering on Pd（111）

1 INTRODUCTION The interaction of water molecules with metal sur- faces plays a vital role in a number of important pro- cesses, such as corrosion, heterogeneous catalysis, electrochemical processes in aqueous solutions, hydrogen production, etc.[1] The structure and pro- perties of water adsorbed on well-defined metal sur- faces have been the subject of numerous experi- mental and theoretical investigations. There have been a number of experimental studies of water on metal surfaces throu…     

The inorganic perspective of nerve growth factor: interactions of Cu2+ and Zn2+ with the N-terminus fragment of nerve growth factor encompassing the recognition domain of the TrkA receptor

There is a significant overlap between brain areas with Zn(2+) and Cu(2+) pathological dys-homeostasis and those in which the nerve growth factor (NGF) performs its biological role. The protein NGF is necessary for the development and maintenance of the sympathetic and sensory nervous systems. Its flexible N-terminal region has been shown to be a critical domain for TrkA receptor binding and activation. Computational analyses show that Zn(2+) and Cu(2+) form pentacoordinate complexes involving both the His4 and His8 residues of the N-terminal domain of one monomeric unit and the His84 and Asp105 residues of the other monomeric unit of the NGF active dimer. To date, neither experimental data on the coordination features have been reported, nor has one of the hypotheses according to which Zn(2+) and Cu(2+) may have different binding environments or the Ser1 α-amino group could be involved in coordination been supported. The peptide fragment, encompassing the 1-14 sequence of the human NGF amino-terminal domain (NGF(1-14)), blocked at the C terminus, was synthesised and its Cu(2+) and Zn(2+) complexes characterized by means of potentiometric and spectroscopic (UV/Vis, CD, NMR, and EPR) techniques. The N-terminus-acetylated form of NGF(1-14) was also investigated to evaluate the involvement of the Ser1 α-amino group in metal-ion coordination. Our results demonstrate that the amino group is the first anchoring site for Cu(2+) and is involved in Zn(2+) coordination at physiological pH. Finally, a synergic proliferative activity of both NGF(1-14) and the whole protein on SHSY5Y neuroblastoma cell line was found after treatment in the presence of Cu(2+). This effect was not observed after treatment with the N-acetylated peptide fragment, demonstrating a functional involvement of the N-terminal amino group in metal binding and peptide activity.     

Coexistence of water dimer and hexamer clusters in 3D metal-organic framework structures of Ce(III) and Pr(III) with pyridine-2,6-dicarboxylic acid

Ce(NO3)3.6H2O or Pr(NO3)3.6H2O and pyridine-2,6-dicarboxylic acid form a linear coordination polymeric structure under hydrothermal conditions. Hexameric water clusters join these linear chains through bonding to the metal ions. Other coordinated water and the carboxylate oxygen form an intricate array of hydrogen bonding resulting in a 3D network where each metal ion shows 9-coordination with an approximate D3 symmetry. Dimeric water clusters are also located in the void spaces. In the structure containing Pr(III), the water dimers are hydrogen-bonded to the hexamers, whereas in the Ce(III) structure, the dimers and the hexamers are far apart.     

Properties of GST-CALM expressed in E. coli

Clathrin-coated vesicles (CCVs) are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. CCVs are composed of clathrin and assembly proteins. The clathrin assembly protein lymphoid myeloid leukemia (CALM) gene, encodes a homologoue of the neuronal clathrin assembly protein AP180. In this study, we characterized the properties of the CALM expressed in E. coli. The molecular weight of bacterially expressed GST-CALM fusion protein was approximately 105 kD on SDS-PAGE. The CALM protein could promote clathrin triskelia into clathrin cages and could bind the preformed clathrin cage. However, 33 kD N-terminal domain of CALM could not bind pre-assembled clathrin cages, but assemble clathrin triskelia into clathrin cages. The CALM protein was bound to SH3 domain through N-terminal domain1, in vitro. The CALM protein is proteolyzed by caspase 3, caspase 8 and calpain through C-terminal domain.     

Influence of anions in silver supramolecular frameworks: structural characteristics and sorption properties

The complexation of a preorganized thioether-functionalized bis(pyrazolyl)methane ligand (L) with silver precursors produces supramolecular structures organized at two hierarchical levels: [AgL](6)(X)(6) metal-organic cyclic hexamers and their organization in 3D architectures. The cyclic toroidal hexamers of 22-26 ? external diameter are found to be stable already in solution before self-assembly into the crystalline state. In the 3D lattice, the hexameric building block are arranged in different highly symmetric space groups as a function of a variety of anions (cubic Fd3 with PF(6)(-) or BF(4)(-) and rhombohedral R3 with CF(3)SO(3)(-) or NO(3)(-)) and form cavities with the geometrical shapes of Platonic solids (tetrahedron and octahedron) that can be occupied by a variety of solvent molecules. Upon evacuation, cubic crystals can produce stable frameworks with permanent porosity, which can absorb reversibly several vapors, CO(2) and CH(4).     

Molecular encapsulation in pyrogallolarene hexamers under nonequilibrium conditions

Pyrogallol[4]arene is a macrocycle with a concave surface and 12 peripheral hydroxyl groups that mediate its self-assembly to form hexamers of octahedral symmetry in the solid state, in solution, and in the gas phase. These hexamers enclose approximately 1300 ?(3) of space, which is filled with small molecules. In this study, we show that solvent-free conditions for guest entrapment in these hexamers, using molten guest molecules as solvent and allowing the capsules to assemble during cooling, results in exceptionally kinetically stable encapsulation complexes that are not formed in the presence of solvent and are not thermodynamically stable. The capsules' kinetic stabilities are strongly dependent on the size and shape of both guest and solvent molecules, with larger or nonplanar molecules with rigid geometries providing enhanced stability. The greatest observed barrier to guest exchange, ΔG(?) = 32 ± 0.7 kcal mol(-1) for encapsulated CCl(4) → encapsulated pyrene, is, to the best of our knowledge, indicative of the most powerful kinetic trap ever observed for a synthetic, hydrogen-bonded encapsulation complex. Detailed NMR studies of the structures of the assemblies and the kinetics and mechanisms for guest exchange reveal that subtle differences in guest and solvent structure can impart profound effects on the behavior of the systems. Kinetic and thermodynamic stability, capsule symmetry and structure, guest tumbling rates, susceptibility to disruption by polar solvents, and even the mechanism for equilibration-the presence or absence of supramolecular intermediates-are all greatly influenced. The strongest observed kinetic traps provide encapsulation complexes that are not at equilibrium but are nonetheless indefinitely persistent at ambient temperatures, a property that invites future applications of supramolecular chemistry in open systems where equilibrium is not possible.     

The octarepeat domain of the prion protein binds Cu(II) with three distinct coordination modes at pH 7.4

The prion protein (PrP) binds Cu2+ in its N-terminal octarepeat domain. This unusual domain is comprised of four or more tandem repeats of the fundamental sequence PHGGGWGQ. Previous work from our laboratories demonstrates that at full copper occupancy, each HGGGW segment binds a single Cu2+. However, several recent studies suggest that low copper occupancy favors different coordination modes, possibly involving imidazoles from histidines in adjacent octapeptide segments. This is investigated here using a combination of X-band EPR, S-band EPR, and ESEEM, along with a library of modified peptides designed to favor different coordination interactions. At pH 7.4, three distinct coordination modes are identified. Each mode is fully characterized to reveal a series of copper-dependent octarepeat domain structures. Multiple His coordination is clearly identified at low copper stoichiometry. In addition, EPR detected copper-copper interactions at full occupancy suggest that the octarepeat domain partially collapses, perhaps stabilizing this specific binding mode and facilitating cooperative copper uptake. This work provides the first complete characterization of all dominant copper coordination modes at pH 7.4.     

Differential isotope dansylation labeling combined with liquid chromatography mass spectrometry for quantification of intact and N-terminal truncated proteins

The N-terminal amino acids of proteins are important structure units for maintaining the biological function, localization, and interaction networks of proteins. Under different biological conditions, one or several N-terminal amino acids could be cleaved from an intact protein due to processes, such as proteolysis, resulting in the change of protein properties. Thus, the ability to quantify the N-terminal truncated forms of proteins is of great importance, particularly in the area of development and production of protein-based drugs where the relative quantity of the intact protein and its truncated form needs to be monitored. In this work, we describe a rapid method for absolute quantification of protein mixtures containing intact and N-terminal truncated proteins. This method is based on dansylation labeling of the N-terminal amino acids of proteins, followed by microwave-assisted acid hydrolysis of the proteins into amino acids. It is shown that dansyl labeled amino acids are stable in acidic conditions and can be quantified by liquid chromatography mass spectrometry (LC–MS) with the use of isotope analog standards.     

Spectral Features of Canthaxanthin in HCP2. A QM/MM Approach

The increased interest in sequencing cyanobacterial genomes has allowed the identification of new homologs to both the N-terminal domain (NTD) and C-terminal domain (CTD) of the Orange Carotenoid Protein (OCP). The N-terminal domain homologs are known as Helical Carotenoid Proteins (HCPs). Although some of these paralogs have been reported to act as singlet oxygen quenchers, their distinct functional roles remain unclear. One of these paralogs (HCP2) exclusively binds canthaxanthin (CAN) and its crystal structure has been recently characterized. Its absorption spectrum is significantly red-shifted, in comparison to the protein in solution, due to a dimerization where the two carotenoids are closely placed, favoring an electronic coupling interaction. Both the crystal and solution spectra are red-shifted by more than 50 nm when compared to canthaxanthin in solution. Using molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) studies of HCP2, we aim to simulate these shifts as well as obtain insight into the environmental and coupling effects of carotenoid–protein interactions.     

NanoESI Mass Spectrometry of Rubisco and Rubisco Activase Structures and Their Interactions with Nucleotides and Sugar Phosphates

Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is the protein that is responsible for the fixation of carbon dioxide in photosynthesis. Inhibitory sugar phosphate molecules, which can include its substrate ribulose-1,5-bisphosphate (RuBP), can bind to Rubisco catalytic sites and inhibit catalysis. These are removed by interaction with Rubisco activase (RA) via an ATP hydrolytic reaction. Here we show the first nanoESI mass spectra of the hexadecameric Rubisco and of RA from a higher plant (tobacco). The spectra of recombinant, purified RA revealed polydispersity in its oligomeric forms (up to hexamer) and that ADP was bound. ADP was removed by dialysis against a high ionic strength solution and nucleotide binding experiments showed that ADP bound more tightly to RA than AMP-PNP (a non-hydrolysable ATP analog). There was evidence that there may be two nucleotide binding sites per RA monomer. The oligomerization capacity of mutant and wild-type tobacco RA up to hexamers is analogous to the subunit stoichiometry for other AAA+ enzymes. This suggests assembly of RA into hexamers is likely the most active conformation for removing inhibitory sugar phosphate molecules from Rubisco to enable its catalytic competency. Stoichiometric binding of RuBP or carboxyarabinitol bisphosphate (CABP) to each of the eight catalytic sites of Rubisco was observed.     

The role of intrinsically disordered regions in the structure and functioning of small heat shock proteins

Small heat shock proteins (sHsp) form a large ubiquitous family of proteins expressed in all phyla of living organisms. The members of this family have low molecular masses (13-43 kDa) and contain a conservative α-crystallin domain. This domain (about 90 residues) consists of several β-strands forming two β-sheets packed in immunoglobulinlike manner. The α-crystallin domain plays an important role in formation of stable sHsp dimers, which are the building blocks of the large sHsp oligomers. A large N-terminal domain and a short C-terminal extension flank the α-crystallin domain. Both the N-terminal domain and the C-terminal extension are flexible, susceptible to proteolysis, prone to posttranslational modifications, and are predominantly intrinsically disordered. Differently oriented N-terminal domains interact with each other, with the core α-crystallin domain of the same or neighboring dimers and play important role in formation of large sHsp oligomers. Phosphorylation of certain sites in the N-terminal domain affects the sHsp quaternary structure, the sHsp interaction with target proteins and the sHsp chaperone-like activity. The C-terminal extension often carrying the conservative tripeptide (I/V/L)-X-(I/V/L) is capable of binding to a hydrophobic groove on the surface of the core α-crystallin domain of neighboring dimer, thus affecting the plasticity and the overall structure of sHsp oligomers. The Cterminal extension interacts with target proteins and affects their interaction with the α-crystallin domain increasing solubility of the complexes formed by sHsp and their targets. Thus, disordered N- and C-terminal sequences play important role in the structure, regulation and functioning of sHsp.     

The influence of mobile phase demixion on thin-layer chromatographic enantioseparation of ibuprofen and naproxen

In our earlier article we presented the results of tracing the enantioseparation of the two test analytes (ibuprofen and naproxen) by means of video densitometry and scanning densitometry. In that way we demonstrated an excellent performance of this combined approach to the thin-layer chromatographic detection in the area of enantioseparation. In this paper we study an impact of the four different mobile phases on the enantioseparation of the scalemic mixtures of ibuprofen and naproxen on the silica gel layers impregnated with L-arginine as chiral selector. The main component of all the investigated mobile phases is 2-propanol. Mobile phase 1 consists of pure 2-propanol, while mobile phases 2-4 contain, respectively, ca. 0.66, 1.32, and 1.98 g/L of glacial acetic acid in 2-propanol. Acetic acid is used to protonate L-arginine, as the involved retention mechanism consists of the ion pair formation between L-arginine in the cationic form and the chiral 2-arylpropionic acids (2-APAs), ibuprofen and naproxen, in the anionic form. It is shown that in the absence of glacial acetic acid no enantioseparation can be obtained. Then with adding of 0.66 g/L glacial acetic acid partial enantioseparation of the naproxen and ibuprofen antimers is obtained, with a simultaneous effect of the mobile phase demixion. With the amount of acetic acid increasing, the effect of demixion becomes increasingly perceptible. In that case the displacement effect is observed (and mathematically modeled), which results in compressing of the antimer pairs by the second front of mobile phase. The obtained results allow a deeper insight into the mechanism of enantioseparation with the two test 2-APAs. A combined impact of the crystalline chirality of silica gel and the molecular chirality of L-arginine on the vertical and the horizontal enantioseparation of ibuprofen and naproxen is also discussed.     

Inactivation of microbial arginine deiminases by L-canavanine

Arginine deiminase (ADI) catalyzes the hydrolytic conversion of L-arginine to ammonia and L-citrulline as part of the energy-producing L-arginine degradation pathway. The chemical mechanism for ADI catalysis involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate. The structure of the Pseudomonas aeruginosa ADI-(L-arginine) complex guided the design of arginine analogs that might react with the ADIs to form inactive covalent adducts during catalytic turnover. One such candidate is L-canavanine, in which an N-methylene of L-arginine is replaced by an N-O. This substance was shown to be a slow substrate-producing O-ureido-L-homoserine. An in depth kinetic and mass spectrometric analysis of P. aeruginosa ADI inhibition by L-canavanine showed that two competing pathways are followed that branch at the Cys-alkylthiouronium ion intermediate. One pathway leads to direct formation of O-ureido-L-homoserine via a reactive thiouronium intermediate. The other pathway leads to an inactive form of the enzyme, which was shown by chemical model and mass spectrometric studies to be a Cys-alkylisothiourea adduct. This adduct undergoes slow hydrolysis to form O-ureido-L-homoserine and regenerated enzyme. In contrast, kinetic and mass spectrometric investigations demonstrate that the Cys-alkylthiouronium ion intermediate formed in the reaction of L-canavanine with Bacillus cereus ADI partitions between the product forming pathway (O-ureido-L-homoserine and free enzyme) and an inactivation pathway that leads to a stable Cys-alkylthiocarbamate adduct. The ADIs from Escherichia coli, Burkholderia mallei, and Giardia intestinalis were examined in order to demonstrate the generality of the L-canavanine slow substrate inhibition and to distinguish the kinetic behavior that defines the irreversible inhibition observed with the B. cereus ADI from the time controlled inhibition observed with the P. aeruginosa, E. coli, B. mallei, and G. intestinalis ADIs.     

Inherent flexibility and protein function: The open/closed conformational transition in the N-terminal domain of calmodulin

The key to understand a protein's function often lies in its conformational dynamics. We develop a coarse-grained variational model to investigate the interplay between structural transitions, conformational flexibility, and function of the N-terminal calmodulin domain (nCaM). In this model, two energy basins corresponding to the "closed" apo conformation and "open" holo conformation of nCaM are coupled by a uniform interpolation parameter. The resulting detailed transition route from our model is largely consistent with the recently proposed EFbeta-scaffold mechanism in EF-hand family proteins. We find that the N-terminal parts of the calcium binding loops shows higher flexibility than the C-terminal parts which form this EFbeta-scaffold structure. The structural transition of binding loops I and II are compared in detail. Our model predicts that binding loop II, with higher flexibility and earlier structural change than binding loop I, dominates the open/closed conformational transition in nCaM.     

Functional Analysis of the Extended N-Terminal Region in PLC-δ1 (MlPLC-δ1) from the Mud Loach,Misgurnus mizolepis

Mud loach phospholipase C-δ1 (MlPLC-δ1) contains all the characteristic domains found in mammalian PLC-δ isozymes (pleckstrin homology domain, EF-hands, X–Y catalytic region, and C2 domain) as well as an extended 26-amino acid (aa)-long N-terminal region that is an alternative splice form of PLC-δ1 and is novel to vertebrate PLC-δ. In the present structure–function analysis, deletion of the extended N-terminal region caused complete loss of phosphatidylinositol (PI)- and phosphatidylinositol 4,5-bisphosphate (PIP₂)-hydrolyzing activity in MlPLC-δ1. Additionally, recombinant full-length MlPLC-δ1 PLC activity was reduced in a dose-dependent manner by coincubation with the 26-aa protein fragment. Using a protein–lipid overlay assay, both full-length MlPLC-δ1 and the 26-aa protein fragment had substantial affinity for PIP₂, whereas deletion of the 26-aa region from MlPLC-δ1 (MlPLC-δ1-deletion) resulted in lower affinity for PIP₂. These results suggest that the novel N-terminal exon of MlPLC-δ1 could play an important role in the regulation of PLC-δ1.