Dissociation energies of deoxyribose nucleotide dimer anions measured using blackbody infrared radiative dissociation.
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The dissociation kinetics of deprotonated deoxyribose nucleotide dimers were measured using blackbody infrared radiative dissociation. Experiments were performed with noncovalently bound dimers of phosphate, adenosine (dAMP), cytosine (dCMP), guanosine (dGMP), thymidine (dTMP), and the mixed dimers dAMP.dTMP and dGMP.dCMP. The nucleotide dimers fragment through two parallel pathways, resulting in formation of the individual nucleotide or nucleotide + HPO3 ion. Master equation modeling of this kinetic data was used to determine threshold dissociation energies. The dissociation energy of (dGMP.dCMP-H)- is much higher than that for the other nucleotide dimers. This indicates that there is a strong interaction between the nucleobases in this dimer, consistent with the existence of Watson-Crick hydrogen bonding between the base pairs. Molecular mechanics simulations indicate that Watson-Crick hydrogen bonding occurs in the lowest energy structures of (dGMP.dCMP-H)-, but not in (dAMP.dTMP-H)-. The trend in gas phase dissociation energies is similar to the trend in binding energies measured in nonaqueous solutions within experimental error. Finally, the acidity ordering of the nucleotides is determined to be dTMP < dGMP < dCMP < dAMP, where dAMP has the highest acidity (largest delta Gacid). (+info)
Electrospray ionization mass spectrometric analysis of microcystins, cyclic heptapeptide hepatotoxins: modulation of charge states and [M + H]+ to [M + Na]+ ratio.
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Electrospray ionization mass spectrometry was used to develop a rapid, sensitive, and accurate method for determination and identification of hepatotoxic microcystins, cyanobacterial cyclic heptapeptides. To optimize the electrospray ionization conditions, factors affecting charge state distribution, such as amino acid components of sample, proton affinity of the additives, and additive concentration, were investigated in detail and a method for controlling charge states was developed to provide molecular-related ions for assignment of molecular weight and reasonably abundant precursor ions for MS/MS analysis. A procedure for identification of microcystins consisting of known amino acids was proposed: for microcystins giving abundant [M + 2H]2+ ions, the addition of nitrogen-containing bases to the aqueous sample solution is effective to obtain an increased intensity of [M + H]+ ions, whereas the addition of Lewis acids containing nitrogen can produce increased abundances of [M + 2H]2+ ions for microcystins giving weak [M + 2H]2+ ions. Microcystins possessing no arginine residue always give sodium adduct ions [M + Na]+ as the base peak, and these are difficult to fragment via low energy collision-induced dissociation to yield structurally informative products; the addition of oxalic acid increases [M + H]+ ion abundances, and these fragment readily. (+info)
Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.
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The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment. (+info)
Isolation and characterization of hydrophobic polypeptides in human bile.
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Polypeptides were isolated from human bile by extraction with chloroform/methanol, followed by reversed-phase chromatography in methanol/ethylene chloride and gel filtration in chloroform/methanol. Peptides were characterized by SDS/PAGE, sequence analysis and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. This identified haemoglobin alpha chain, ATP synthase lipid-binding protein subunit 9, an N-terminal fragment of mac25/insulin-like growth factor-binding protein 7 and an internal fragment of monocyte differentiation antigen CD14, all not described previously in bile. In addition, alpha1-antitrypsin, known in bile from previous work, was also identified. The hydrophobic character of haemoglobin alpha chain is not apparent from its amino acid sequence, but the other polypeptides all have major hydrophobic segments. These results show that several proteins are removed upon organic solvent extraction used for delipidation during the preparation of samples for proteome analysis. Several of the polypeptides found are unexpectedly present in bile, suggesting that specific excretion mechanisms may be involved. (+info)
Ozone model for bonding of an O2 to heme in oxyhemoglobin.
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Several rather different models of the Fe-o2 bond in oxyhemoglobin have previously been proposed, none of which provide a satisfactory explanation of several properties. We propose a new model for the bonding of an O2 to the Fe of myoglobin and hemoglobin and report ab initio generalized valence bond and configuration interaction calculations on FeO2 that corroborate this model. Our model is based closely upon the bonding in ozone which recent theoretical studies have shown to be basically a biradical with a singlet state stabilized by a three-center four-electron pi bond. In this model, the facile formation and dissociation of the Fe-O2 bond is easily rationalized since the O2 always retains its triplet ground state character. The ozone model leads naturally to a large negative electric field gradient (in agreement with Mossbauer studies) and to z-polarized (perpendicular to the heme) charge transfer transitions. It also suggests that the 1.3 eV transition, present in HbO2 and absent in HbCO, is due to a porphyrin-to-Fe transition, analogous to that of ferric hemoglobins (e.g., HbCN). (+info)
The physical characteristics of neodymium iron boron magnets for tooth extrusion.
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Impaction and non-eruption of teeth is a common problem encountered in orthodontics and many techniques have been proposed for the management of this condition. It has been advocated that a system utilizing magnets would supply a continuous, directionally sensitive, extrusive force, through closed mucosa and thus provide not only a physiological sound basis for successful treatment, but also reduce the need for patient compliance and appliance adjustment. This ex vivo investigation examined in detail the physical characteristics of neodymium iron boron magnets employed in attraction in order to assess their usefulness in the clinical situation. Attractive force and magnetic flux density measurements were recorded for nine sets of magnet pairs with differing morphologies. The effect of spatial relationship on force was assessed by varying vertical, transverse and horizontal positions of the magnets relative to each other, and by altering the pole face angles. The data obtained suggest that magnets with larger pole face areas and longer magnetic axes provide the best performance with respect to clinical usefulness. It was possible to formulate a specific relationship between force and flux density for each magnet pair. This relationship can be used in the clinical management of unerupted teeth to predict the force between the magnets by measuring the magnetic flux density present at mucosal level. The results indicate that magnetic systems may, indeed, have a place in the treatment of unerupted teeth. (+info)
Interaction with GroEL destabilises non-amphiphilic secondary structure in a peptide.
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The Escherichia coli molecular chaperone GroEL can functionally interact with non-native forms of many proteins. An inherent property of non-native proteins is the exposure of hydrophobic residues and the presence of secondary structure elements. Whether GroEL unfolds or stabilises these structural elements in protein substrates as a result of binding has been the subject of extended debate in the literature. Based on our studies of model peptides of pre-formed helical structure, we conclude that the final state of a GroEL-bound substrate is dependent on the conformational flexibility of the substrate protein and the distribution of hydrophobic residues, with optimal association when these are able to present a cluster of hydrophobic residues in the binding interface. (+info)
Catalytic properties and conformation of hydrophobized alpha-chymotrypsin incorporated into a bilayer lipid membrane.
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A set of artificially hydrophobized alpha-chymotrypsin derivatives, carrying 2-11 stearoyl residues per enzyme molecule, were synthesized and their catalytic parameters and conformation in water solution and in the liposome-bound state were investigated. Hydrophobization of alpha-chymotrypsin and its further incorporation into phosphatidylcholine (PC) liposomes have no effect on the rate constant of the N-acetyl-L-tyrosine ethyl ester (ATEE) ester bond hydrolysis (k(cat)). At the same time, an increase in the number of stearoyl residues attached to the enzyme results in a drastic decrease of ATEE binding to the active center (K(M) increase). Incorporation of the hydrophobized enzyme into the PC liposome membrane results in K(M) recovery to nearly that of native alpha-chymotrypsin. The above changes are accompanied by partial unfolding of the enzyme molecules observed by fluorescence measurements. The obtained results are of interest to mimic the contribution of surface hydrophobic sites in the functioning of membrane proteins. (+info)