The role of tandem acyl carrier protein domains in polyunsaturated fatty acid biosynthesis

Acyl carrier protein (ACP) plays an essential role in fatty acid and polyketide biosynthesis, and most of the fatty acid synthases (FASs) and polyketide synthases (PKSs) known to date are characterized with a single ACP for each cycle of chain elongation. Polyunsaturated fatty acid (PUFA) biosynthesis is catalyzed by the PUFA synthase, and all PUFA synthases known to date contain tandem ACPs (ranging from 5 to 9). Using the Pfa PUFA synthase from Shewanella japonica as a model system, we report here that these tandem ACPs are functionally equivalent regardless of their physical location within the PUFA synthase subunit, but the total number of ACPs controls the overall PUFA titer. These findings set the stage to interrogate other domains and subunits of PUFA synthase for their roles in controlling the final PUFA products and could potentially be exploited to improve PUFA production.     

Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine candidates

The introduction of glycoconjugate vaccines marks an important point in the fight against various infectious diseases. The covalent conjugation of relevant polysaccharide antigens to immunogenic carrier proteins enables the induction of a long-lasting and robust IgG antibody response, which is not observed for pure polysaccharide vaccines. Although there has been remarkable progress in the development of glycoconjugate vaccines, many crucial parameters remain poorly understood. In particular, the influence of the conjugation site and strategy on the immunogenic properties of the final glycoconjugate vaccine is the focus of intense research. Here, we present a comparison of two cysteine selective conjugation strategies, elucidating the impact of both modifications on the structural integrity of the carrier protein, as well as on the immunogenic properties of the resulting glycoconjugate vaccine candidates. Our work suggests that conjugation chemistries impairing structurally relevant elements of the protein carrier, such as disulfide bonds, can have a dramatic effect on protein immunogenicity.

The introduction of glycoconjugate vaccines marks an important point in the fight against various infectious diseases.     

AquaBridge: A novel method for systematic search of structural water molecules within the protein active sites

We have developed a novel method for calculation of the water bridges that can be formed in the active sites of proteins in the absence or in the presence of small‐molecule ligands. We tested its efficiency on a representative set of human ATP‐binding proteins, and show that the docking accuracy of ligands can be substantially improved when water bridges are included in the modeling of protein–ligand interactions. Our analysis of binding pocket hydration can be a useful addition to the in silico approaches of Drug Design. © 2015 Wiley Periodicals, Inc.     

The role of the protein matrix in green fluorescent protein fluorescence

In the ground state of the highly conjugated green fluorescent protein (GFP), the chromophore should be planar. However, numerous crystal structures of GFP and GFP-like proteins have been reported with slightly twisted chromophores. We have previously shown that the protein cavity surrounding the chromophore in wild-type GFP is not complementary with a planar chromophore. This study shows that the crystal structure of wild-type GFP is not an anomaly: most of the GFP and GFP-like proteins in the protein databank have a protein matrix that is not complementary with a planar chromophore. When the pi-conjugation across the ethylenic bridge of the chromophore is removed the protein matrix will significantly twist the freely rotating chromophore from the relatively planar structures found in the crystal structures. The possible consequences of this nonplanar deformation on the photophysics of GFP are discussed. A volume analysis of the cis-trans-isomerization of HBDI, a GFP chromophore model compound, reveals that its hula-twist motion is volume conserving. This means that, if the GFP chromophore or GFP chromophore model compounds undergo a cis-trans-isomerization in a volume-constricting medium, such as a protein matrix or viscous liquid, it will probably isomerize by means of a HT-type motion.     

A carrier protein strategy yields the structure of dalbavancin

Many large natural product antibiotics act by specifically binding and sequestering target molecules found on bacterial cells. We have developed a new strategy to expedite the structural analysis of such antibiotic-target complexes, in which we covalently link the target molecules to carrier proteins, and then crystallize the entire carrier-target-antibiotic complex. Using native chemical ligation, we have linked the Lys-D-Ala-D-Ala binding epitope for glycopeptide antibiotics to three different carrier proteins. We show that recognition of this peptide by multiple antibiotics is not compromised by the presence of the carrier protein partner, and use this approach to determine the first-ever crystal structure for the new therapeutic dalbavancin. We also report the first crystal structure of an asymmetric ristocetin antibiotic dimer, as well as the structure of vancomycin bound to a carrier-target fusion. The dalbavancin structure reveals an antibiotic molecule that has closed around its binding partner; it also suggests mechanisms by which the drug can enhance its half-life by binding to serum proteins, and be targeted to bacterial membranes. Notably, the carrier protein approach is not limited to peptide ligands such as Lys-D-Ala-D-Ala, but is applicable to a diverse range of targets. This strategy is likely to yield structural insights that accelerate new therapeutic development.     

The role of neutral defects in the structural chemistry of liquid water

For defective water associates with an extra hydrogen atom (n-defective associates), size and structure effects on ionization potentials compared to defectless (normal) associates of similar structures have been investigated by quantum chemical and molecular mechanics methods. The ionization potentials of small n defective water associates increase (from fractions of eV to 7 eV–8 eV) with the number of water molecules in the associate. These are most probably the source of a hydrated electron that are responsible for the ensuing equilibrium between all defects in liquid water: neutral n and p defects and ion defects (H _aq ⁺ , OH _aq ^– , e _aq ^– ). Delocalization of an odd electron in defective associates stabilizes the latter and promotes their recombinations, forming hydrated water molecules, hydrogen peroxide, and gaseous hydrogen. Structural instability of fullerene (H₂O)₂₀ relative to normal associates has been found. This compound is stabilized by the endo inclusion of a hydrogen atom, and exo fixation of the hydrogen atom gives rise to an extra source of hydrated electrons.Original Russian Text Copyright © 2004 by G. A. Domrachev, D. A. Selivanovskii, E. G. Domracheva, L. G. Domracheva, A. I. Lazarev, P. A. Stunzhas, S. F. Shishkanov, and V. L. VaksTranslated from Zhurnal Strukturnoi Khimii, Vol. 45, No. 4, pp. 670–677, July–August, 2004.This revised version was published online in April 2005 with a corrected cover date.     

Hydrolytically active tetranuclear nickel complexes with structural resemblance to the active site of urease

Reaction of the new asymmetric ligand 2-(N-isopropyl-N-((1-methylimidazolyl)methyl)aminomethyl)-6-(N-carboxylmethyl-N-((1-methylimidazolyl)methyl) aminomethyl)-4-methylphenol (ICIMP) with nickel perchlorate and diphenylacetic acid leads to the formation of tetranuclear nickel complexes, whose crystal structures reveal that they consist of dimers of dimers in which each Ni(2) unit has a coordination environment that is similar to the active site of urease. One complex has been shown to coordinate urea and catalyze the hydrolysis of an organophosphate monoester.     

Comparison of active and passive environmental sampling for safeguards applications

Towards development of electrical conductivity based sensors for online detection of formation of third phase during reprocessing of Pu rich spent nuclear fuels, laboratory studies were carried out using U⁴⁺ solutions in various experimental conditions. Third phases were generated by extracting U⁴⁺ from nitric acid medium by 1.09 M TBP at different A/O (A: aqueous, O: organic) ratios. The results of this study indicate that the third phase is nearly 100–300 times more conducting than lean organic phase and saturated phase. The higher conductivity of third phase as compared to that of other two phases is explained based on the principle of reverse micelle formation and charge movement between the micro emulsion globules by percolation phenomenon.     

Oscillatory passive active transition during the corrosion in nickel chromium layer systems

The electrochemical corrosion of the porous layer system nickel/chromium was investigated combining classical open circuit measurements, cyclic voltammetry, and zero-resistance Ammeter experiments. Under moderate conditions as there are low chloride concentration, pH 6, and high oxygen content the nickel layer acts as sacrificial anode, whereas at the chromium top layer oxygen reduction takes place. Under conditions of limited access of oxygen into pores potential oscillations occur which indicates a change of corrosion mechanism. Thereby the anode reaction changes between nickel and chromium dissolution. The respective cathode reaction is oxygen reduction or hydrogen evolution. The modeling of the potential oscillation reveals the character of the potential-dependent Flade potential as function of the proton concentration.     

The putative lead-binding carrier protein in rat epidermis using210Pb

To verify the usefulness of hair as an indicator of²¹⁰Pb and¹⁰⁹Cd accumulation in rats their distribution in different organs were determined. Whereas the radioactivity following²¹⁰Pb i.p. administration after seven days is extremely diminished to compare with that after two days, a considerable accumulation with time of¹⁰⁹Cd was observed under similar experimental conditions. The binding of lead in rat epidermis and hair was investigated and relatively tightly bound²¹⁰Pb to extracted proteins was established. By electrophoretic analysis of epidermal proteins using PAGE-SDS binding of radiolead to a protein carrier with m.w. of about 16 000 was detected. The work was performed in the frame of IAEA Coordinated Research Programme /RC 3673/RB/.     

Investigation of the role of the carrier gas in capillary gas-solid chromatography

Nonideal interactions of the sorbate and the carrier gas and adsorption of the sorbate on the adsorbent surface in capillary gas-solid chromatography were studied. Chromatographic retention was found to be largely determined by adsorption processes. With respect to the retention coefficients (capacity factors) of a sorbate (k) with different carrier gases (P₁ and P₂), the correlation relationshipk(P₂) =A·k(P₁) +B (A, B are parameters of the equation) is closely obeyed. The advantages of carbon dioxide as the carrier gas were analyzed; the use of carbon dioxide allows the efficiency of the column to be enhanced.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 627–633, March, 1996.     

The role of the active site tyrosine in the mechanism of lytic polysaccharide monooxygenase

Catalytic breakdown of polysaccharides can be achieved more efficiently by means of the enzymes lytic polysaccharide monooxygenases (LPMOs). However, the LPMO mechanism has remained controversial, preventing full exploitation of their potential. One of the controversies has centered around an active site tyrosine, present in most LPMO classes. Recent investigations have for the first time obtained direct (spectroscopic) evidence for the possibility of chemical modification of this tyrosine. However, the spectroscopic features obtained in the different investigations are remarkably different, with absorption maximum at 420 and 490 nm, respectively. In this paper we use density functional theory (DFT) in a QM/MM formulation to reconcile these (apparently) conflicting results. By modeling the spectroscopy as well as the underlying reaction mechanism we can show how formation of two isomers (both involving deprotonation of tyrosine) explains the difference in the observed spectroscopic features. Both isomers have a [TyrO–Cu–OH]⁺ moiety with the OH in either the cis- or trans-position to a deprotonated tyrosine. Although the cis-[TyrO–Cu–OH]⁺ moiety is well positioned for oxidation of the substrate, preliminary calculations with the substrate reveal that the reactivity is at best moderate, making a protective role of tyrosine more likely.

With QM/MM, we investigate the mechanism of tyrosine deprotonation in lytic polysaccharide monooxygenases. Our results support deprotonation and our calculated UV-vis spectra show that two isomers must be formed to match recent experiments.     

The influence of mechanical activation and the nature of a carrier on the structural and diffusion properties of zeolite-containing catalysts

The influence of mechanical activation and the nature of a carrier on the structural and diffusion properties of zeolite-containing catalysts, including the microporous and mesoporous structure of zeolites, was studied. Zeolite-matrix contacts were found to provide the accessibility of the inside volume of zeolite pores. In addition, new pores of a larger size appeared. The IR spectra of adsorbed molecules were used to determine the effective diffusion coefficients of methanol in the porous system of zeolites. It was shown that there was some optimum zeolite pore radius at which the largest diffusion coefficient was attained.     

Processing and characterization of new passive and active oxysulfide glasses in the Ge-Ga-Sb-S-O system

New passive and active oxysulfide glasses have been prepared in the Ge-Ga-Sb-S-O system employing a two-step melting process which involves the processing of the chalcogenide glass (ChG) and its subsequent melting with amorphous GeO₂ or Sb₂O₃ powder. Optical characterization of the oxysulfide glasses has shown that the UV cut-off wavelength decreases with increasing oxygen content. Using Raman spectroscopy, correlations have been made between the formation of new Ge- and Sb-based oxysulfide structural units, the Sb content and the O/S ratio. We demonstrate the successful processing of active rare earth doped oxysulfide glasses by melting the chalcogenide glass with Er₂O₃ and Sb₂O₃ and also by melting the Er³⁺ doped chalcogenide glass with Sb₂O₃. Modification of the emission spectra at 1500 nm of Er³⁺ doped samples with the introduction of oxygen revealed that Er³⁺ most likely exists in dual O- and S-neighbor environments. Finally, the photo-response of these new glasses upon near-IR femtosecond laser irradiation has been investigated as a function of Sb content and O/S ratio and shows that the glasses are photo-sensitive to NIR fs laser light with the magnitude of the photo-sensitivity dependent on the glass’ Sb content and O/S ratio.     

Exploring the use of a structural alphabet for structural prediction of protein loops

 The prediction of loop conformations is one of the challenging problems of homology modeling, owing to the large sequence variability associated with these parts of protein structures. In the present study, we introduce a search procedure that evolves in a structural alphabet space deduced from a hidden Markov model to simplify the structural information. It uses a Bayesian criterion to predict, from the amino acid sequence of a loop region, its corresponding word in the structural alphabet space. The results show that our approach ranks 30% of the target words with the best score, 50% within the five best scores. Interestingly, our approach is also suited to accept or not the prediction performed. This allows the ranking of 57% of the target words with the best score, 67% within the five best scores, accepting 16% of learned words and rejecting 93% of unknown words. Received: 17 July 2000 / Accepted: 5 January 2001 / Published online: 3 April 2001     

The importance of short structural motifs in protein structure analysis

Summary Proteins tend to use recurrent structural motifs on all levels of organization. In this paper we first survey the topics of recurrent motifs on the local secondary structure level and on the global fold level. Then, we focus on the intermediate level which we call the short structural motifs. We were able to identify a set of structural building blocks that are very common in protein structure. We suggest that these building blocks can be used as an important link between the primary sequence and the tertiary structure. In this framework, we present our latest results on the structural variability of the extended strand motifs. We show that extended strands can be divided into three distinct structural classes, each with its own sequence specificity. Other approaches to the study of short structural motifs are reviewed.