Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein-surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein-surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein-surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein-surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.     

Interaction of giant extracellular Glossoscolex paulistus hemoglobin (HbGp) with ionic surfactants: a MALDI-TOF-MS study

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by approximately 144 subunits containing heme groups with molecular masses in the range of 16-19kDa forming a monomer (d) and a trimer (abc), and around 36 non-heme structures, named linkers (L). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS) analysis was performed recently, to obtain directly information on the molecular masses of the different subunits from HbGp in the oxy-form. This technique demonstrated structural similarity between HbGp and the widely studied hemoglobin of Lumbricus terrestris (HbLt). Indeed, two major isoforms (d(1) and d(2)) of identical proportions with masses of 16,355+/-25 and 16,428+/-24Da, respectively, and two minor isoforms (d(3) and d(4)) with masses around 16.6kDa were detected for monomer d of HbGp. In the present work, the effects of anionic sodium dodecyl sulfate (SDS) and cationic cethyltrimethylammonium chloride (CTAC) on the oligomeric structure of HbGp have been studied by MALDI-TOF-MS in order to evaluate the interaction between ionic surfactants and HbGp. The data obtained with this technique show an effective interaction of cationic surfactant CTAC with the two isoforms of monomer d, d(1) and d(2), both in the whole protein as well as in the pure isolated monomer. The results show that up to 10 molecules of CTAC are bound to each isoform of the monomer. Differently, the mass spectra obtained for SDS-HbGp system showed that the addition of the anionic surfactant SDS does not originate any mass increment of the monomeric subunits, indicating that SDS-HbGp interaction is, probably, significantly less effective as compared to CTAC-HbGp one. The acid pI of the protein around 5.5 is, probably, responsible for this behavior. The results of this work suggest also some interaction of both surfactants with linker chains as well as with trimers, as judged from observed mass increments. Our data are consistent with a recent spectroscopic study showing a strong interaction between CTAC and HbGp at physiological pH [P.S.Santiago, et al, Biochim. Biophys. Acta 1770 (2007) 506-517.].     

Autoxidation studies of extracellular hemoglobin of Glossoscolex paulistus at pH 9: cyanide and hydroxyl effect

The complex oligomeric assembly of the hemoglobin subunits may influence the autoxidation rate. To understand this relation, the rate of autoxidation was studied at pH 9.0, where the Glossoscolex paulistus Hemoglobin (GpHb) dissociates. At alkaline pH, this hemoglobin is dissociated into monomers, trimers and tetramers, allowing the study of the integral protein and monomer subunit autoxidation on independent experiments. The autoxidation rate was evaluated in the presence and absence of cyanide (CN(-)), a strong field ligand to the ferric ion. The oxidation kinetic was monitored using the UV-vis absorption at 415 nm, and resulted in: i) bi-exponential kinetics for the whole hemoglobin (indicating a fast and a slow oxidative process) and ii) mono-exponential for the monomer (indicating a single process). To understand the specific characteristics of each autoxidation process, Arrhenius plots allowed the determination of the activation energy. The experimental results indicate for the whole hemoglobin in the absence of CN(-) an activation energy of 150 +/- 10 kJ mol(-1) for the fast and the slow processes. Under the same conditions the monomer displayed an activation energy of 160 +/- 10 kJ mol(-1), very close to the value obtained for the integral protein. The pseudo-second order rate constant for the whole protein autoxidation by CN(-) showed two different behaviors characterized by a rate constant k(CN1)' = 0.11 +/- 0.02 s(-1) mol(-1) L for CN(-) concentrations lower than 0.012 mol L(-1); and k(CN1)" = 0.76 +/- 0.04 s(-1) mol(-1) L at higher concentrations for the fast process, while the slow process remain constant with k(CN2) = 0.033 +/- 0.002 s(-1) mol(-1) L. The monomer has a characteristic rate constant of 0.041 +/- 0.002 s(-1) mol(-1) L for all cyanide concentrations. Comparing the results for the slow process of the whole hemoglobin and the oxidation of the monomer, it is possible to infer that the slow process has a strong contribution of the monomer in the whole hemoglobin kinetic. Moreover, as disulfide linkers sustain the trimer assembly, cooperativity may explain the higher kinetic constant for this subunit.     

Dynamic light scattering and optical absorption spectroscopy study of pH and temperature stabilities of the extracellular hemoglobin of Glossoscolex paulistus

The extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted of subunits containing heme groups, monomers and trimers, and nonheme structures, called linkers, and the whole protein has a minimum molecular mass near 3.1 × 10⁶ Da. This and other proteins of the same family are useful model systems for developing blood substitutes due to their extracellular nature, large size, and resistance to oxidation. HbGp samples were studied by dynamic light scattering (DLS). In the pH range 6.0-8.0, HbGp is stable and has a monodisperse size distribution with a z-average hydrodynamic diameter (D_h) of 27 ± 1 nm. A more alkaline pH induced an irreversible dissociation process, resulting in a smaller D_h of 10 ± 1 nm. The decrease in D_h suggests a complete hemoglobin dissociation. Gel filtration chromatography was used to show unequivocally the oligomeric dissociation observed at alkaline pH. At pH 9.0, the dissociation kinetics is slow, taking a minimum of 24 h to be completed. Dissociation rate constants progressively increase at higher pH, becoming, at pH 10.5, not detectable by DLS. Protein temperature stability was also pH-dependent. Melting curves for HbGp showed oligomeric dissociation and protein denaturation as a function of pH. Dissociation temperatures were lower at higher pH. Kinetic studies were also performed using ultraviolet-visible absorption at the Soret band. Optical absorption monitors the hemoglobin autoxidation while DLS gives information regarding particle size changes in the process of protein dissociation. Absorption was analyzed at different pH values in the range 9.0-9.8 and at two temperatures, 25°C and 38°C. At 25°C, for pH 9.0 and 9.3, the kinetics monitored by ultraviolet-visible absorption presents a monoexponential behavior, whereas for pH 9.6 and 9.8, a biexponential behavior was observed, consistent with heme heterogeneity at more alkaline pH. The kinetics at 38°C is faster than that at 25°C and is biexponential in the whole pH range. DLS dissociation rates are faster than the autoxidation dissociation rates at 25°C. Autoxidation and dissociation processes are intimately related, so that oligomeric protein dissociation promotes the increase of autoxidation rate and vice versa. The effect of dissociation is to change the kinetic character of the autoxidation of hemes from monoexponential to biexponential, whereas the reverse change is not as effective. This work shows that DLS can be used to follow, quantitatively and in real time, the kinetics of changes in the oligomerization of biologic complex supramolecular systems. Such information is relevant for the development of mimetic systems to be used as blood substitutes.     

Glossoscolex paulistus extracellular hemoglobin (HbGp) oligomeric dissociation upon interaction with sodium dodecyl sulfate: Isothermal titration calorimetry (ITC)

Annelid erythrocruorins are respiratory proteins with high cooperativity and low autoxidation rates. The giant extracellular hemoglobin of the earthworm, Glossoscolex paulistus (HbGp), has a molecular mass of 3.6 MDa. In this work, isothermal titration calorimetry (ITC), together with DLS and fluorescence emission have been used to investigate the interaction of SDS with the HbGp in the oxy‐form, at pH 7.0. Our ITC and DLS results show that addition of SDS induces oxy‐HbGp oligomeric dissociation, while a small amount of protein aggregation is observed only by DLS. Moreover, the oligomeric dissociation process is favored at lower protein concentrations. The temperature effect does not influence significantly the interaction of SDS with the hemoglobin, due to the similarities presented by the critical aggregation concentration (cac) and critical micelle concentration (cmc′) for the mixtures. The increase of oxy‐HbGp concentration leads to a slight variation of the cac values for the SDS‐oxy‐HbGp mixture, attributed mainly to the noncooperative electrostatic binding of surfactant to protein. However, the cmc′ values increase considerably, associated to a more cooperative hydrophobic binding. Complementary pyrene fluorescence emission studies show formation of pre‐micellar structures of the mixture already at lower SDS concentrations. This study opens the possibility of the evaluation of the surfactant effect on the hemoglobin stability by ITC, which is made for the first time with this extracellular hemoglobin. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1065–1076, 2014.     

On the stability of the extracellular hemoglobin of Glossoscolex paulistus, in two iron oxidation states, in the presence of urea

The stability of the Glossoscolex paulistus hemoglobin (HbGp), in two iron oxidation states (and three forms), as monitored by optical absorption, fluorescence emission and circular dichroism (CD) spectroscopies, in the presence of the chaotropic agent urea, is studied. HbGp oligomeric dissociation, denaturation and iron oxidation are observed. CD data show that the cyanomet-HbGp is more stable than the oxy-form. Oxy- and cyanomet-HbGp show good fits on the basis of a two state model with critical urea concentrations at 220-222 nm of 5.1±0.2 and 6.1±0.1 mol/L, respectively. The three-state model was able to reveal a subtle second transition at lower urea concentration (1.0-2.0 mol/L) associated to partial oligomeric dissociation. The intermediate state for oxy- and cyanomet-HbGp is very similar to the native state. For met-HbGp, a different equilibrium, in the presence of urea, is observed. A sharp transition at 1.95±0.05 mol/L of denaturant is observed, associated to oligomeric dissociation and hemichrome formation. In this case, analysis by a three-state model reveals the great similarity between the intermediate and the unfolded states. Analysis of spectroscopic data, by two-state and three-state models, reveals consistency of obtained thermodynamic parameters for HbGp urea denaturation.     

Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.     

Ferric species of the giant extracellular hemoglobin of Glossoscolex paulistus as function of pH: an EPR study on the irreversibility of the heme transitions

The present article is focused on the transitions of ferric heme species of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) induced by successive alterations in pH, involving alkaline and acid mediums. Electron paramagnetic resonance (EPR) is the spectroscopy used to evaluate the transitions that occur in the first coordination sphere of ferric ion as a consequence of ligand changes in a wide range of pH, since this tool is very sensitive to slight changes that occur in the heme pocket of paramagnetic species. This approach is adequate to obtain information regarding the reversibility/irreversibility that involves the heme transitions induced by pH, since the degree of reversibility is associated to the intensity of the changes that occur in the spatial configuration of the polypeptide chains, which is clearly associated to the first coordination sphere. The results demonstrate a significant degree of irreversibility of heme transitions, since the final species, which do not present any change after 6 h of its respective formations, are quite different of the initial species. The results denote that the more stable species are the bis-histidine (hemichrome) and pentacoordinate species, due to the properties of their ligands and to the mechanical influence of the respective subunits. EPR spectra allow to distinguish the types of hemichrome species, depending on the reciprocal orientation between the histidine axial ligands, in agreement with Walker's Classification [Walker, F.A., 1999. Magnetic spectroscopic (EPR, ESEEM, M?ssbauer, MCD and NMR) studies of low-spin ferriheme centers and their corresponding heme proteins. Coord. Chem. Rev. 185-186, 471-534]. However, these transitions are not completed, i.e., the appearance of a determined species does not mean the total consumption of its precursor species, implying the coexistence of several types of species, depending on pH. Furthermore, it is possible to conclude that a "pure" EPR spectrum of aquomet ferric species is an important indicator of a high level of conservation referent to the "native" configuration of whole hemoglobin, which is only encountered at pH 7.0. The results allow to infer important physico-chemical properties as well as to evaluate aspects of the structure-activity relationship of this hemoprotein, furnishing information with respect to the denaturation mechanism induced by drastic changes in pH. These data are very useful since HbGp has been proposed as prototype of substitute of blood, thus requiring wide knowledge about its structural and chemical properties.     

Small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) studies of amide phospholipids

Varying chemically the structure of phospholipids in the region between hydrophobic and hydrophilic segments is expected to have a strong influence on the interaction with water and the phase behavior. This is studied in this work with the motivation to investigate these lipids as potential inhibitors of phospholipase A2. Thus the amide phospholipids L-ether-amide-PC (1-O-hexadecyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine), L-ester-amide-PC (1-palmitoyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine) and L-ether-amide-PE (1-O-hexadecyl-2-N-palmitoyl-2-deoxy-sn-glycero-3-phosphoethanolamine) have been synthesized and characterized. The phase behavior and thermal transitions in buffer dispersions are examined by a combination of high-sensitivity differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS) experiments between 10 and 80 degrees C at pH 8.9. The onset temperatures determined from DSC measurements agree well with the starting temperatures of changes in the repeat distance obtained by SAXS measurements. The phases observed are lamellar both below and above the main phase transition. The phase transition temperatures and enthalpies depend strongly on the substitutions in sn-1 position and head group structure. The lamellar repeat distance in gel and liquid-crystalline phases increases with increasing temperature for L-ester-amide-PC and L-ether-amide-PC, whereas the temperature dependence is opposite for the L-ether-amide-PE. The observed behavior is discussed and compared with that of DPPC and DPPE, indicating the strong dependence of hydration and phase behavior on head group structure.     

The effect of iron binding on the conformation of transferrin. A small angle x-ray scattering study.

Distance distribution functions, p(r), radii of gyration, Rg, and radii of gyration of cross section, Rq, of apotransferrin, monoferric transferrin, and diferric transferrin have been compared. The alteration of Rg and Rq upon iron binding has been determined by a difference method. An unusual feature of the stepwise structural changes of transferrin upon iron saturation is that binding of the first ferric ion is responsible for more than half of the whole change in Rq, whereas Rg alters significantly only after the binding of the second ferric ion.     

Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein–surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein–surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein–surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein–surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.     

Structure of Bombyx mori silk fibroin before spinning in solid state studied with wide angle x-ray scattering and (13)C cross-polarization/magic angle spinning NMR

The structure of a crystalline form of Bombyx mori silk fibroin, commonly found before the spinning process (known as silk I), has been proposed as a repeated beta-turn type II-like structure by combining data obtained from solid-state two dimensional spin-diffusion nuclear magnetic resonance and rotational-echo double-resonance (T. Asakura et al., J Mol Biol, in press). In this paper, the WAXS pattern of alanine-glycine alternating copolypeptide, (Ala-Gly)(15) with silk I form which was used for a silk I model of B. mori silk fibroin was observed. The pattern calculated with the silk I model proposed by us is well reproduced the observed one, indicating the validity of the proposed silk I model. In addition, two peptides of the other repeated sequences which contain Tyr or Val residues in the silk fibroin,23 were synthesized; (Ala-Gly-Tyr-Gly-Ala-Gly)(5) and (X-Gly)(15) where X is Tyr for the 7th, 15th and 23th residues, and Val for the 11th residue and Ala for other residues. There are no sharp peaks in the WAXS patterns, and therefore both samples are in the non-crystalline state. This is in agreement with the (13)C CP/MAS NMR result, where the conformation is mainly random coil.     

Small angle X-ray scattering study of meso-tetrakis (4-sulfonatophenyl) porphyrin in aqueous solution: a self-aggregation model

The aggregate morphology of meso-tetrakis(4-sulfonatophenyl) porphyrin (TPPS(4)) in aqueous solution is investigated by using small angle x-ray scattering (SAXS) technique. Measurements were performed at pH 4.0 and 9.0 to monitor the pH influence on the structural parameters of the aggregates. Radii of gyration were obtained from distance distribution functions p(r) analysis. The experimental data of TPPS(4) at pH 4.0 showed well-defined oscillations characteristic of large aggregates in contrast to the SAXS curve of 5 mM TPPS(4) at pH 9.0, where both a significant decrease in the intensity and the disappearance of the oscillation peaks suggest the dissociation of the aggregate. A 340-A long "hollow" cylinder with shell thickness of 20 A, compatible to the porphyrin molecule dimension, represents well the scattering curve of the aggregates at pH 4.0. According to the fitting parameters, 26 porphyrin molecules self-associate into a ringlike configuration in the plane of the cylinder cross-section. The total number of porphyrin molecules in the whole aggregate was also estimated as approximately 3000. The model compatible to SAXS data of a hollow cylinder with J-aggregation in the cross-section and H-aggregation (columnar stacking) between the cylinder layers is consistent with optical absorption spectroscopic data both in the literature and obtained in this work.     

Physicochemical study of floranol, its copper(II) and iron(III) complexes, and their inhibitory effect on LDL oxidation

The antioxidant activity of floranol (3,5,7,2'-tetrahydroxy-6-methoxy-8-prenylflavanone), a new flavonoid isolated from the roots of Dioclea grandiflora, was evaluated by the inhibition of human low-density lipoprotein (LDL) oxidation. Floranol increased its oxidation lag-phase significantly in a dose-dependent manner. As the antioxidant mechanism may involve metal coordination, we have undertaken a detailed study of floranol interactions with Cu(II) and Fe(III) by combination of UV-visible (UV-Vis) and mass spectrometries and cyclic voltammetry. The acidity constants of the ligand as well as the stability constants of the metal complexes were calculated. The pKa values of 6.58, 11.97 and 13.87 were determined and the following acidity order is proposed 7-OH>5-OH>2'-OH. The best fit between experimental and calculated spectra was obtained assuming the formation of two Cu(II) complexes: [CuL] logbeta=19.34+/-0.05 and [CuL(2)](2-) logbeta=26.4+/-0.10 and three Fe(III) complexes: [FeL(3)](3-) logbeta=44.72+/-0.09, [FeL(2)](-) logbeta=35.32+/-0.08 and [FeL](+) logbeta=19.51+/-0.04. In addition, copper and iron reduction is less favorable in the presence of floranol. These results indicate that floranol can efficiently bind Cu(II) and Fe(III) ions thus preventing their effect on LDL oxidation.     

Cation Modulation of Hemoglobin Interaction with Sodium n-Dodecyl Sulfate (SDS). III: Calcium Interaction with R- and Mixed Spin States of Hemoglobin S at pH 5.0: The Musical Chair Paradox

We investigate the interaction of Ca²⁺ (0–500 µM) and a membrane mimic (0.60 mM SDS) with both the R- and mixed spin states hemoglobin S (HbS) as a function of time. These interactions were carried out at pH 5.0. We aim at ascertaining if there is or are differences in the UV–Visible spectra of such interactions to account for the dynamics of calcium ion concentrations [Ca²⁺] in initiating structures which may ultimately suggest HbS polymerization and or resistance to Plasmodium attack. From our results, we conclude that (a) simultaneous interaction of 40 µM Ca²⁺ and 0.60 mM SDS with the R state protein would promote structural formations that can “lock up” the protein for nucleation on the membranes and or become cytotoxic to the parasite; (b) simultaneous R state HbS-SDS or R state HbS-Ca²⁺ would lead to enhanced hemin formation and less deoxyHb species. This condition is unlikely to precipitate polymerization in the HbS but the resulting hemin would poison the parasite; (c) the mixed spin state HbS-SDS and 40 µM Ca²⁺ interaction yields more toxic products to that of the interaction of the mixed spin HbS-SDS with 500 µM Ca²⁺ thus suggesting why the 40 µM Ca²⁺ is important in parasite Hb proteolysis; and (d) pronounced structural changes on interaction with SDS and Ca²⁺ are more in the R state to the mixed spin state.     

X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution

Crystallography supplies unparalleled detail on structural information critical for mechanistic analyses; however, it is restricted to describing low energy conformations of macromolecules within crystal lattices. Small angle X-ray scattering (SAXS) offers complementary information about macromolecular folding, unfolding, aggregation, extended conformations, flexibly linked domains, shape, conformation, and assembly state in solution, albeit at the lower resolution range of about 50 A to 10 A resolution, but without the size limitations inherent in NMR and electron microscopy studies. Together these techniques can allow multi-scale modeling to create complete and accurate images of macromolecules for modeling allosteric mechanisms, supramolecular complexes, and dynamic molecular machines acting in diverse processes ranging from eukaryotic DNA replication, recombination and repair to microbial membrane secretion and assembly systems. This review addresses both theoretical and practical concepts, concerns and considerations for using these techniques in conjunction with computational methods to productively combine solution scattering data with high-resolution structures. Detailed aspects of SAXS experimental results are considered with a focus on data interpretation tools suitable to model protein and nucleic acid macromolecular structures, including membrane protein, RNA, DNA, and protein-nucleic acid complexes. The methods discussed provide the basis to examine molecular interactions in solution and to study macromolecular flexibility and conformational changes that have become increasingly relevant for accurate understanding, simulation, and prediction of mechanisms in structural cell biology and nanotechnology.     

Influence of extracellular pH on the cytotoxicity, cellular accumulation, and DNA interaction of novel pH-sensitive 2-aminoalcoholatoplatinum(II) complexes

Extracellular acidity is a frequent pathophysiological condition of solid tumors offering possibilities for improving the tumor selectivity of molecular therapy. This might be accomplished by prodrugs with low systemic toxicity, attaining their full antitumor potency only under acidic conditions, such as bis(2-aminoalcoholato-κ²N,O)platinum(II) complexes that are activated by protonation of alcoholato oxygen, resulting in cleavage of platinum–oxygen bonds. In this work, we examined whether the pH dependency of such compounds is reflected in differential biological activity in vitro. In particular, the pH dependence of cytotoxicity, cellular accumulation, DNA platination, GMP binding, effects on DNA secondary structure, cell cycle alterations, and induction of apoptosis was investigated. Enhanced cytotoxicity of five of these complexes in non-small-cell lung cancer (A549) and colon carcinoma (HT-29) cells at pH 6.0 in comparison with pH 7.4 was confirmed: 50 % growth inhibition concentrations ranged from 42 to 214 μM in A549 cells and from 35 to 87 μM in HT-29 cells at pH 7.4 and decreased at pH 6.0 to 11–50 and 7.3–25 μM, respectively. The effects induced by all five pH-sensitive compounds involve increased 5′-GMP binding, cellular accumulation, and DNA platination as well as stronger effects on DNA secondary structure at pH 6.0 than at pH 7.4. As exemplified by treatment of A549 cells with a 2-amino-4-methyl-1-pentanolato complex, induction of apoptosis is enhanced at pH 6.5. These results confirm the increased reactivity and in vitro activity of these compounds under slightly acidic conditions, encouraging further evaluation of ring-closed aminoalcoholatoplatinum(II) derivatives in solid tumors in vivo.     

Magic angle spinning NMR study of interaction of N-terminal sequence of dermorphin (Tyr-d-Ala-Phe-Gly) with phospholipids

Two modifications of the Tyr-d-Ala-Phe-Gly tetrapeptide with different C-terminal groups (Tyr-d-Ala-Phe-Gly-OH 1 and Tyr-d-Ala-Phe-Gly-NH(2)2) were investigated by various nuclear magnetic resonance sequences under magic angle spinning. The structural constraints obtained from the magic angle spinning nuclear magnetic resonance measurements suggest that both peptides are aligned on the surface of the membrane and that the sandwich-like π-CH(3)-π arrangement of the pharmacophore is preserved. The influence of the chemical modification of the C-terminal residue of 1 and 2 on their interaction with phosphate group of the phospholipid in the subgel phase L(c) and the conformation of the peptides in the liquid crystalline phase L(α) are discussed. The correlation between the X-ray structure of 1 in the solid state and 1 embedded into a membrane in the L(c) phase is presented on the basis of the comparative analysis of the two-dimensional (13)C-(13)C dipolar-assisted rotational resonance cross-peaks and the (13)C isotropic chemical shifts.