Circular Dichroism and Fluorescence Studies of Polyomavirus Major Capsid Protein VP1

The conformational changes of polymavirus (Py) major capsid protein VP₁ in solution by the solution pH, addition of calcium, and ionic strength were examined by circular dichroism (CD) and fluorescence spectroscopy. Comparison of the predicted secondary structures of PyVP₁ and simian virus (SV) 40 by the methods of Chou-Fasman, Gamier et al., and Yang method are presented. Hydropathicity, surface probability, and chain flexibility of PyVP₁ were computer-analyzed by the methods of Kyte and Doolittle, Emini et al., and Karplus and Schulz, respectively. The CD measurements indicate that the secondary structure of PyVP₁ is little dependent on its concentration, Ca²⁺ concentration, and ionic strength, but is strongly pH dependent. Fluorescence studies showed that emission spectra of PyVP₁ are also pH-dependent. At extreme acidic and alkaline pH, the fluorescence intensity of PyVP₁ is decreased and the emission maximum is red-shifted. The fluorescence of PyVP₁ is quenched by the presence of CsCl, KI, and acrylamide. The analyses of the modified Stern–Volmer plots indicate that five of seven tryptophan residues in PyVP₁ are located on the surface of the protein, among which two are accessible to Cs⁺ and the other three are accessible to I^?. The two others are buried more deeply in the interior of the protein molecule.     

Biotin binding changes the conformation and decreases tryptophan accessibility of streptavidin

Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20–28% of the initial fluorescence with (K_SV+V)0.55 M^–1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka=5 M^–1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weberet al., 1989).     

Self-aggregation of a recombinant form of the propeptide NH<Subscript>2</Subscript>-terminal of the precursor of pulmonary surfactant protein SP-B: a conformational study

A recombinant form of the peptide N-terminally positioned from proSP-B (SP-B_N) has been produced in Escherichia coli as fusion with the Maltose Binding Protein, separated from it by Factor Xa cleavage and purified thereafter. This protein module is thought to control assembly of mature SP-B, a protein essential for respiration, in pulmonary surfactant as it progress through the progressively acidified secretory pathway of pneumocytes. Self-aggregation studies of the recombinant propeptide have been carried out as the pH of the medium evolved from neutral to moderately acid, again to neutral and finally basic. The profile of aggregation versus subsequent changes in pH showed differences depending on the ionic strength of the medium, low or moderate, and the presence of additives such as L-arginine (a known aggregation suppressor) and Ficoll 70 (a macromolecular crowder). Circular dichroism studies of SP-B_N samples along the aggregation process showed a decrease in α-helical content and a concomitant increase in β-sheet. Intrinsic fluorescence emission of SP-B_N was dominated by the emission of Trp residues in neutral medium, being its emission maximum shifted to red at low pH, suggesting that the protein undergoes a pH-dependent conformational change that increases the exposure of their Trp to the environment. A marked increase in the fluorescence emission of the extrinsic probe bis-ANS indicated the exposure of hydrophobic regions of SP-B_N at pH 5. The fluorescence of bis-ANS decreased slightly at low ionic strength, but to a great extent at moderate ionic strength when the pH was reversed to neutrality, suggesting that self-aggregation properties of the SP-B_N module could be tightly modulated by the conditions of pH and the ionic environment encountered by pulmonary surfactant during assembly and secretion.     

Exogenous Ubiquinol Analogues Affect the Fluorescence of NCD-4 Bound to Aspartate-160 of Yeast Cytochrome b

Previously, we reported that the carboxyl-reacting reagent DCCD, and its fluorescent derivative NCD-4 binds covalently to aspartate-160 localized in amphipathic helix cd of the CD loop connecting membrane-spanning helices C and D of cytochrome b (Wang et al., 1995). We have investigated the fluorescent properties of NCD-4 to probe possible changes in the cd helix resulting from the binding of exogenous ubiquinol analogues to the bc ₁ complex. Preincubation of the bc ₁ complex with the reduced substrate analogues, DQH₂, DBH₂, and Q₆H₂ resulted in 20–40% increase in the fluorescence emission intensity of NCD-4 and a 10–20% increase in the binding of [¹⁴C]DCCD to the bc ₁ complex. By contrast, preincubation with the oxidized analogues DQ, DB, and Q₆ resulted in a 20–40% decrease in the fluorescence emission intensity of NCD-4 and a 20–40% decrease in the binding of [¹⁴C]DCCD to the bc ₁ complex. Moreover, addition of the reduced ubiquinols to the bc ₁ complex preincubated with NCD-4 resulted in a blue shift in the fluorescence emission spectrum. In addition, incubation of the cytochrome bc ₁ complex reconstituted into proteoliposomes with both reduced and oxidized ubiquinol analogues resulted in changes in the quenching of NCD-4 fluorescence by CAT-16, the spin-label probe that intercalates at the membrane surface. These results indicate that the addition of exogenous ubiquinol to the bc ₁ complex may result in changes in the cd helix leading to a more hydrophobic environment surrounding the NCD-4 binding site. By contrast, preincubation with the inhibitors of electron transfer through the bc ₁ complex had no effect on the binding of NCD-4 to the bc ₁ complex or on the fluorescent emission spectra, which suggests that the binding of the inhibitors does not result in changes in the environment of the NCD-4 binding site.     

Analysis of the Properties of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Insecticidal Toxins Using a Potential-sensitive Fluorescent Probe

A potential-sensitive fluorescent probe, 3,3-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K⁺ ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta. Fluorescence levels were strongly influenced by the pH and ionic strength of the media. Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength. Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane. Valinomycin greatly increased the potential generated by the diffusion of K⁺ ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels. In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

A differential molecualr topography of the Pr and Pfr forms of native oat phytochrome as probed by fluoresence quenching

Tryptophan (Trp) surface topography of the red- and far-red-absorbing forms of phytochrome (Pr, Pfr) ofAvena sativa L. has been investigated by analyzing quenching of the two components of Trp fluorescence decay, in order to understand the differences in the two forms at the molecular level. Stern-Volmer kinetic analysis of the quenching data for two cationic surface quenchers, Cs⁺ and Tl⁺, showed strong quenching of the short component of the Pr fluorescence (Stern-Volmer constants,K _sv , 27.2 and 21.4 M⁻¹, respectively) relative to that of Pfr fluorescenceK _sv , 10.4 and 12.3 M⁻¹, respectively). The long component of the Trp fluorescence was quenched differentially by Cs⁺ and Tl⁺, withK _sv of 9.0 and 19.8 M⁻¹, respectively, for the Pr fluorescence andK _sv of 13.7 and 8.7 M⁻¹, respectively, for the Pfr fluorescence. The results indicate that the phytochrome Trp residues with short fluorescence lifetime are more accessible to the cationic surface quenchers than those with long fluorescence lifetime. The data, taken together with our earlier study (Singh et al. 1988, Biochim, Biophys. Acta936, 395–405), indicate that most, if not all the ten Trp residues of phytochrome, are fluorescent and exist in distinct groups differing in their topography and microenvironment, and the peptide segment containing Trp-774 and Trp-778 within the 55-kilodalton C-terminal domain of phytochrome also undergoes a subtle alteration in its surface topography during Pr→Pfr phototransformation. This paper is dedicated to Professor Hans Mohr in commemoration of his 60th birthday     

The quenching characteristics of potassium iridic chloride and their meaning for the origin of chlorophyll fluorescence components

To understand the origins of the different lifetime components of photosystem 2 (PS2) chlorophyll (Chl) fluorescence we have studied their susceptibility to potassium iridic chloride (K₂IrCl₆) which has been shown to bleach antenna pigments of photosynthetic bacteria (Loach et al. 1963). The addition of K₂IrCl₆ to PS2 particles gives rise to a preferential quenching of the variable Chl fluorescence (F_v). At concentrations lower than 20 M, this is brought about mainly by a decrease in the yield, but not in the lifetime, of the slowest component when all the PS2 reaction centres are closed (F_M). The yield of the middle and fast decays are not significantly altered. This type of quenching is not seen with DNB. The iridate-induced quenching of the initial fluorescence level (F₀) is due to a proportional decrease in the yield and lifetime of the three components and correlates with the observed modification in the relative quantum yield of oxygen evolution. In this concentration range a bleaching of Chl a is seen. At higher iridate levels, greater than 20 M, a proportional decrease in the lifetimes and yields of the three kinetic components is seen at F_M. These changes are associated with a carotenoid bleaching. In isolated light harvesting Chl a/b complexes of PS2 (LHC2), iridate addition converts a 4 ns decay into a 200 ps emission and both types of bleaching are observed. By also measuring the rate of PS2 trap closure versus iridate concentration, we have discussed the results in terms of excitation energy transfer.Abbreviations DNB m-dinitrobenzene - F_M maximum Chl fluorescence - F₀ initial fluorescence - F_v variable fluorescence - I pheophytin a primary electron acceptor of PS2 - P₆₈₀ chlorophyll a of photochemical centre - PS2 photosystem 2 - Q_A primary stable electron acceptor of PS2 - Chl chlorophyll - LHC2 light harvesting Chl a/b complex of PS2 - MES 2(N-morpholino) ethanesulfonic acid - DCMU 3-(3-4-dichlorophenyl) 1-1 dimethylurea - PPBQ phenyl-p-benzo-quinone - BBY PS2-enriched membranes prepared as in Berthold et al. (1981) - Q₄₀₀ PS2 electron acceptor with a midpoint potential of 400 mV     

Role of Intracellular Ca2+ Levels in the Regulation of CD11a/CD18 Mediated Cell Adhesion

CD2, CD3, and MHC class II have been demonstrated to stimulate lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) mediated adhesion (Van Kooyk et al., 1989, Dustin and Springer, 1989; Mourad et al., 1990). Activation of LFA-1 may be mediated by different intracellular signals generated from these stimuli, since previous findings suggest that triggering of LFA-1 through CD2 or CD3 leads to sustained and transient cell adhesion respectively (Van Kooyk et al., 1989). We investigated the role of intracellular signalling pathways in more detail. The results demonstrate that, in addition to protein tyrosine kinase (PTK) and protein kinase C (PKC) mediated signalling, increase in cytosolic-free calcium ([Ca²⁺]_i) levels play a major role in the activation of LFA-1. The calcium iono-phore Ionomycin, which increases [Ca²⁺]_i is capable of directly activating LFA-1. Furthermore, activation of LFA-1 by triggering through CD2, CD3 or MHC class II is associated with an increase in [Ca²⁺]_i levels, with kinetics that directly correlate with cell adhesiveness. Moreover, entry of extracellular Ca²⁺ via Ca-channels is involved in both the CD3-and MHC class II, as well as part of the CD2 induced LFA-1 activation. Depletion of intracellular calcium results in unresponsiveness of LFA-1 to these stimuli, further demonstrating a regulatory role for [Ca²⁺]_i in LFA-1 mediated adhesion.     

Adsorption Mechanism of Cr(VI) onto Coir Pith

Reductive adsorption of Cr(VI) on coir pith (hereafter CP) was examined as a function of pH, ionic strength, and temperature. The CP contains 1.33 meq g^{? 1} phenolic, 0.43 meq g^{? 1} of lactonic, and 0.35 meq g^{? 1} carboxylic sites. Thus the CP surface is enriched with electron-donating oxygen functionalities. As evidenced by infrared (IR) spectroscopy, the Cr(VI) → Cr(III) conversion is facilitated by CP sites that are enriched with O─ O functional groups. The adsorption of reduced Cr(VI) was found to occur via C─ O─ functional groups first forming innersphere complexes with the CP surface, yielding keto (> C═ O) groups on the CP surface. The reductive adsorption of Cr(VI) was almost completed within 3 to 4 h, and it was dependent on pH and background ionic strength, yielding the highest monolayer coverage (9.56E-7 mol m^{? 2}) at pH 3.7 in 0.1 M NaNO₃. The Γ_Cr(III) followed the order with respect to the ionic strength: Γ_{0.1 M} > Γ_{0.01 M} > Γ_{0.001 M}. The initial rate constant, k _i , increased with temperature as k _i ^{313 K} > k _i ^{303 K} > k _i ^{293 K} > k _i ^{283 K}.     

Titanium dioxide nanoparticles preferentially bind in subdomains IB,IIA of HSA and minor groove of DNA

Titanium dioxide nanoparticles (TiO₂-NPs) interaction with human serum albumin (HSA) and DNA was studied by UV–visible spectroscopy, spectrofluorescence, circular dichroism (CD), and transmission electron microscopy (TEM) to analyze the binding parameters and protein corona formation. TEM revealed protein corona formation on TiO₂-NPs surface due to adsorption of HSA. Intrinsic fluorescence quenching data suggested significant binding of TiO₂-NPs (avg. size 14.0 nm) with HSA. The Stern–Volmer constant (K_sv) was determined to be 7.6 × 10² M^?1 (r² = 0.98), whereas the binding constant (K_a) and number of binding sites (n) were assessed to be 5.82 × 10² M^?1 and 0.97, respectively. Synchronous fluorescence revealed an apparent decrease in fluorescence intensity with a red shift of 2 nm at Δλ = 15 nm and Δλ = 60 nm. UV–visible analysis also provided the binding constant values for TiO₂-NPs–HSA and TiO₂-NPs-DNA complexes as 2.8 × 10² M^?1 and 5.4 × 10³ M^?1. The CD data demonstrated loss in α-helicity of HSA and transformation into β-sheet, suggesting structural alterations by TiO₂-NPs. The docking analysis of TiO₂-NPs with HSA revealed its preferential binding with aromatic and non-aromatic amino acids in subdomain IIA and IB hydrophobic cavity of HSA. Also, the TiO₂-NPs docking revealed the selective binding with A-T bases in minor groove of DNA.     

Fluorescence of proteins in aqueous neutral salt solutions. I. Influence of anions

The effects of anions of neutral salts on the fluorescence emission of six proteins as well as on tryptophan and tyrosine were studied in relation to the structure of proteins. Most anions are good quenchers of tryptophyl and tyrosyl fluorescence, free or in proteins. The results with tryptophan and tyrosine indicate involvement of a collisional quenching mechanism due to agreement with Stern–Volmer law. The deactivation of fluorescence probably occurs because of the transition from singlet state to triplet state. Lehrer's modification of Stern–Volmer law was applied to proteins. The effective quenching constants ([K_Q]eff) and the fraction of fluorescence available ([f_a]eff) to the quencher are also calculated. In contrast to its effect on tryptophan, CH₃COO^? quenches tyrosyl fluorescence and ClO₄^? does not. The effects on fluorescence of ribonuclease and free tyrosine are similar and without any changes in emission maximum. The anions are divided into three groups based on the effect they have on tryptophan-containing proteins. (1) NO₃^?, NO₂^?, Br^?, and I^? have high [K_Q]eff values and readily quench tryptophyl fluorescence of proteins causing a shift of emission maximum to a shorter wavelength. This change is due to the specific quenching of “exposed” tryptophan residues which are accessible to quenchers and the observed residual fluorescence is from the “buried” tryptophyls. (2) ClO₄^? and SCN^? also quench fluorescence of tryptophan in proteins and have lower ([K_Q]eff) values. In their presence the fluorescence maximum is shifted to a longer wavelength, which indicates the unfolding of a protein with [(f_a)eff] = 1. (3) Cl^?, CH₃COO^?, and SO₄? do not have a direct effect on the fluorescence of tryptophan. Besides the “direct” effects, “indirect” effects on fluorophors in protein are also seen, pointing out that the neutral salts can interact in more than one manner with proteins. The effectiveness of anions in quenching fluorescence of proteins follows similar sequences which almost resemble the Hofmeister series, viz., SO₄⁼, CH₃COO^? ? Cl^? < ClO₄^? < SCN^? < Br^? < I^? < NO₃^? < NO₂^?.     

Ca2+ effects on Fe(II) interactions with Mn-binding sites in Mn-depleted oxygen-evolving complexes of photosystem II and on Fe replacement of Mn in Mn-containing,Ca-depleted complexes

Fe(II) cations bind with high efficiency and specificity at the high-affinity (HA), Mn-binding site (termed the “blocking effect” since Fe blocks further electron donation to the site) of the oxygen-evolving complex (OEC) in Mn-depleted, photosystem II (PSII) membrane fragments (Semin et al. in Biochemistry 41:5854, 2002). Furthermore, Fe(II) cations can substitute for 1 or 2Mn cations (pH dependent) in Ca-depleted PSII membranes (Semin et al. in Journal of Bioenergetics and Biomembranes 48:227, 2016; Semin et al. in Journal of Photochemistry and Photobiology B 178:192, 2018). In the current study, we examined the effect of Ca²⁺ cations on the interaction of Fe(II) ions with Mn-depleted [PSII(-Mn)] and Ca-depleted [PSII(-Ca)] photosystem II membranes. We found that Ca²⁺ cations (about 50 mM) inhibit the light-dependent oxidation of Fe(II) (5 µM) by about 25% in PSII(-Mn) membranes, whereas inhibition of the blocking process is greater at about 40%. Blocking of the HA site by Fe cations also decreases the rate of charge recombination between Q_A^? and Y_Z^?+ from t_1/2?=?30 ms to 46 ms. However, Ca²⁺ does not affect the rate during the blocking process. An Fe(II) cation (20 µM) replaces 1Mn cation in the Mn₄CaO₅ catalytic cluster of PSII(-Ca) membranes at pH 5.7 but 2 Mn cations at pH 6.5. In the presence of Ca²⁺ (10 mM) during the substitution process, Fe(II) is not able to extract Mn at pH 5.7 and extracts only 1Mn at pH 6.5 (instead of two without Ca²⁺). Measurements of fluorescence induction kinetics support these observations. Inhibition of Mn substitution with Fe(II) cations in the OEC only occurs with Ca²⁺ and Sr²⁺ cations, which are also able to restore oxygen evolution in PSII(-Ca) samples. Nonactive cations like La³⁺, Ni²⁺, Cd²⁺, and Mg²⁺ have no influence on the replacement of Mn with Fe. These results show that the location and/or ligand composition of one Mn cation in the Mn₄CaO₅ cluster is strongly affected by calcium depletion or rebinding and that bound calcium affects the redox potential of the extractable Mn4 cation in the OEC, making it resistant to reduction.

     

Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System

The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT₃ receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1. Virus production by in vivo packaging and production of the recombinant protein was scaled up, the latter to a reactor volume of 11.5 l. A Vibromix^TM agitation system was chosen to overcome aggregation problems of BHK cells in suspension. In the process, cells were first grown to a density of 10⁶ cells/ml, the medium was then exchanged with fresh medium and the culture was infected with the recombinant virus at an estimated multiplicity of infection of 30. 24 h post infection we measured an expression level of 3 million functional 5-HT₃ receptors per cell. For harvesting, the cells were pelleted by centrifugation. The receptor protein was purified in a single step (Hovius et al., 1998) by exploiting the hexa-His tag at minimal protein loss (51% yield). Experiments to optimise expression resulted in yields up to 8 million receptors per cell, when the pH of a suspension culture was controlled at pH 7.3. Rapid virus generation and protein production, high protein yields as well as successful large scale application have made the SFV expression system attractive to produce large quantities of recombinant protein in a very short time. After optimisation of the expression conditions (in particular by setting the pH at 7.3), yields were increased twofold.     

The quenching of tryptophan fluorescence by protonated and unprotonated imidazole

The fluorescence life-time of N-acetyl-tryptophan-amide (NATA) was measured by multifrequency phase fluorometry, in the presence of increasing concentrations of imidazole. Two pH values were tested, pH 4.5 where imidazole is fully protonated and pH 9.0 where it is fully unprotonated. At both pH values, the inverse life-time increases in a non-linear way with the imidazole concentration, showing that imidazole is not a high efficiency collisional quencher. The data can be analysed in terms of the formation of a complex with a reduced fluorescence life-time. The rate constants for association (at 25°C) are around 5 (±0.2) × 10⁹ M^–1 s^–1 and are thus diffusion controlled. The association equilibrium constant is strongly pH dependent and is much higher than the expected value of 0.4 M^–1 for a collisional complex. The intrinsic fluorescence life-time of the complex is 1.56 (±0.02) ns at pH 9.0 and 1.82 (±0.03) ns at pH 4.5, as compared to 2.37 (±0.03) ns for free NATA at pH 9.0 and 2.83 (±0.05) at pH 4.5 (all atI = 0.34). This means that at both pH values the fluorescence life-time of NATA in the complex is reduced to 61 (±0.5)% of its value in the free state. Despite this, the protonated form of imidazole is a better quencher at low concentrations, owing to a longer residence-time of the complex. At high viscosity the association equilibration is too slow and the system is described by two life-times. The quenching effect ofHis-18 on the fluorescence of the proximalTrp-94 of barnase (Locwenthal et al. 1991, Willaert et al. 1991) is discussed in terms of these findings.     

Interaction of β‐cyclodextrin‐capped CdSe quantum dots with inorganic anions and cations

A facile method was developed for the preparation of water soluble β‐Cyclodextrin (β‐CD)‐modified CdSe quantum dots (QDs) (β‐CD‐QDs) by directly replacing the oleic acid ligands on the QDs surface with β‐CD in an alkaline aqueous solution. The as‐prepared QDs show good stability in aqueous solution for several months. Oxoanions, including phosphoric acid ion, sulphite acid ion and carbonic acid ion, affect the fluorescence of β‐CD‐QDs. Among them, H₂PO₄^– exhibited the largest quenching effect. For the polyprotic acids (HO)₃AO, the effect of acidic anions on the fluorescence of β‐CD‐QDs was in the order: monoanion (HO)₂AO₂^– > dianion (HO)AO₃^2– >> trianion AO₄^3–. After photoactivation for several days in the presence of anions at alkaline pH, the β‐CD‐QDs exhibited strong fluorescence emission. The effect of various heavy and transition metal ions on the fluorescence properties of the β‐CD‐QDs was investigated further. It was found that Ag⁺, Hg²⁺ and Co²⁺ have significant quenching effect on the fluorescence of the β‐CD‐QDs. The Stern–Volmer quenching constants increased in the order: Hg²⁺ < Co²⁺ <Ag⁺. The adsorption model of metal ions on β‐CD‐QDs was explored. Copyright © 2011 John Wiley & Sons, Ltd.     

1H NMR studies on ferricytochromec 3 fromDesulfovibrio vulgaris Miyazaki F and its interaction with ferredoxin I

Summary The¹H NMR signals of the heme methyl, propionate and related chemical groups of cytochromec ₃ fromDesulfovibrio vulgaris Miyazaki F (D.v. MF) were site-specifically assigned by means of ID NOE, 2D DQFCOSY and 2D TOCSY spectra. They were consistent with the site-specific assignments of the hemes with the highest and second-lowest redox potentials reported by Fan et al. (Biochemistry,29 (1990) 2257–2263). The site-specific heme assignments were also supported by NOE between the methyl groups of these hemes and the side chain of Val¹⁸. All the results contradicted the heme assignments forD.v. MF cytochromec ₃ made on the basis of electron spin resonance (Gayda et al. (1987)FEBS Lett.,217 57–61). Based on these assignments, the interaction of cytochromec ₃ withD.v. MF ferredoxin I was investigated by NMR. The major interaction site of cytochromec ₃ was identified as the heme with the highest redox potential, which is surrounded by the highest density of positive charges. The stoichiometry and association constant were two cytochromec ₃ molecules per monomer of ferredoxin I and 10⁸ M^–2 (at 53 mM ionic strength and 25°C), respectively.     

Interaction of Anions and Cations in Regulating Energy Distribution between the Two Photosystems

Cations such as Mg²⁺ regulate spillover of absorbed excitation energy mainly in favour of photosystem (PS) 2. Effect of low concentration (<10 mM) of the monovalent cation Na⁺ on chlorophyll (Chl) a fluorescence was completely overridden by divalent cation Mg²⁺ (5 mM). Based on Chl a fluorescence yield and 77 K emission measurements, we revealed the role and effectiveness of anions (Cl^-, SO₄ ^2-, PO₄ ^3-) in lowering the Mg²⁺-induced PS2 fluorescence. The higher the valency of the anion, the lesser was the expression of Mg²⁺ effect. Anions may thus overcome Mg²⁺ effects up to certain extent in a valency dependent manner, thereby diverting more energy to PS1 even in the presence of MgCl₂. They may do so by reversing Mg²⁺-induced changes.     

CD-monitored redox titration of the Rieske Fe-S protein of Rhodobacter sphaeroides: pH dependence of the midpoint potential in isolated bc1 complex and in membranes

The redox potential of the Rieske Fe-S protein has been investigated using circular dichroism (CD)-spectroscopy. The CD features characteristic of the purified bc₁ complex and membranes of Rhodobacter sphaeroides were found in the region between 450 and 550 nm. The difference between reduced and oxidized CD-spectra shows a negative band at about 500 nm with a half of width 30 nm that corresponds to the specific dichroic absorption of the reduced Rieske protein (Fee, J.A. et al. (1984) J. Biol. Chem. 259, 124–133; Degli Esposti, M. et al. (1987) Biochem. J. 241, 285–290; Rich, P.R. and Wiggins, T.E. (1992) Biochem. Soc. Trans. 20, 241S). It was found that the redox potential at pH 7.0 for the Rieske center in the isolated bc₁ complex and in chromatophore membranes from the R-26 strain of Rb. sphaeroides is 300±5 mV. In chromatophores from the BC17C strain of Rb. sphaeroides, the E_m value measured for the Rieske iron-sulfur protein (ISP) was higher (315±5 mV), but the presence of carotenoids made measurement less accurate. The E_m varied with pH in the range above pH 7, and the pH dependence was well fit either by one pK at 7.5 in the range of titration, or by two pK values, pK₁=7.6 and pK₂=9.8. Similar titrations and pK values were found for the Rieske Fe-S protein in the isolated bc₁ complex and membranes from the R-26 strain of Rb. sphaeroides. The results are discussed in the context of the mechanism of quinol oxidation by the bc₁ complex, and the role of the iron sulfur protein in formation of a reaction complex at the Q_o-site.     

Structural determinants of the intrinsic fluorescence emission in notexin and phospholipase A2 enzymes

Fluorescence measurements of the homologous proteins, notexin and PLA₂ enzymes fromNaja naja atra, Naja nigricollis, and Hemachatus haemachatus venoms, showed that the wavelength of maximum emission and the quantum yield of their intrinsic fluorescence emission spectra were different. To verify the factors which affected their fluorescence characteristics, the dynamics of tryptophan residues in those homologous proteins were studied by quenching with acrylamide, iodide, and cesium. The degrees of exposure of tryptophanyl groups in notexin and PLA₂ enzymes assessed by acrylamide quenching were found to be the major factor that determined their fluorescence characteristics. However, the positively charged groups surrounding tryptophan residues of PLA₂ enzymes fromN. naja atra andN. nigricollis venoms might affect the quantum yield of their fluorophores. Tryptophan residues of notexin were in an environment with less fluctuation, which did not allow free diffusion of ionic quencher. This might render its typtophan residues to fluoresce at a shorter wavelength. These results suggested that the structural determinants affecting the intrinsic fluorescence emission of homologous proteins can be easily assessed by quenching studies.