Acidic pH triggers conformational changes at the NH2-terminal propeptide of the precursor of pulmonary surfactant protein B to form a coiled coil structure

Pulmonary surfactant protein SP-B is synthesized as a larger precursor, proSP-B. We report that a recombinant form of human SP-B_N forms a coiled coil structure at acidic pH. The protonation of a residue with pK = 4.8 ± 0.06 is the responsible of conformational changes detected by circular dichroism and intrinsic fluorescence emission. Sedimentation velocity analysis showed protein oligomerisation at any pH condition, with an enrichment of the species compatible with a tetramer at acidic pH. Low 2,2,2,-trifluoroethanol concentration promoted β-sheet structures in SP-B_N, which bind Thioflavin T, at acidic pH, whereas it promoted coiled coil structures at neutral pH. The amino acid stretch predicted to form β-sheet parallel association in SP-B_N overlaps with the sequence predicted by several programs to form coiled coil structure. A synthetic peptide (⁶⁰W-E⁸⁵) designed from the sequence of the amino acid stretch of SP-B_N predicted to form coiled coil structure showed random coil conformation at neutral pH but concentration-dependent helical structure at acidic pH. Sedimentation velocity analysis of the peptide indicated monomeric state at neutral pH (s_{20, w} = 0.55 S; Mr ~ 3 kDa) and peptide association (s_{20, w} = 1.735 S; Mr = ~ 14 kDa) at acidic pH, with sedimentation equilibrium fitting to a Monomer-Nmer-Mmer model with N = 6 and M = 4 (Mr = 14692 Da). We propose that protein oligomerisation through coiled-coil motifs could then be a general feature in the assembly of functional units in saposin-like proteins in general and in the organization of SP-B in a functional surfactant, in particular.     

Comparative studies on the properties of the extrinsic manganese-stabilizing protein from higher plants and of a synthetic peptide of its C-terminus

The present study describes a comparative analysis on the fluorescence properties of the manganese-stabilizing protein (MSP), a synthetic peptide corresponding to its C terminus and a 7:1 (molar ratio) mixture of N-acetyl-tyrosine and N-acetyl-tryptophan, respectively, together with reconstitution experiments of oxygen evolution in MSP-depleted photosystem II (PS II) membrane fragments. It is found: (i) at neutral pH, the fluorescence from Trp²⁴¹ is strongly diminished in MSP solutions, whereas it highly dominates the overall emission from the C-terminus peptide; (ii) at alkaline pH, the emission of Tyr and Trp is quenched in both, MSP and C-terminus peptide, with increasing pH but the decline curve is shifted by about two pH units towards the alkaline region in MSP; (iii) a drastically different pattern emerges in the 7:1 mixture where the Trp emission even slightly increases at high pH; (iv) the anisotropy of the fluorescence emission is wavelength-independent (310-395 nm) and indicative of one emitter type (Trp) in the C-terminus peptide and of two emitter types (Tyr, Trp) in MSP; and (v) in MSP-depleted PS II membrane fragments the oxygen evolution is restored (up to 85% of untreated control) by rebinding of MSP but not by the C-terminus peptide, however, the presence of the latter diminishes the restoration effect of MSP. A quenching mechanism of Trp fluorescence by a next neighbored tyrosinate in the peptide chain is proposed and the relevance of the C terminus of MSP briefly discussed.     

Misconceptions over Förster resonance energy transfer between proteins and ANS/bis-ANS: Direct excitation dominates dye fluorescence

Our aim was to disprove the widespread misconception that Förster resonance energy transfer (FRET) is the only explanation for observing fluorescence from ANS (8-anilino-1-naphthalenesulfonic acid) and bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt) following excitation at 280 nm in the presence of protein. From ultraviolet (UV) absorption spectra and fluorescence emission spectra of bis-ANS and ANS in buffer and ethanol, direct excitation at 280 nm was found to be the dominant mechanism for the resulting dye fluorescence. Furthermore, Tyr/Trp quenching studies were performed for solutions of N-acetyl-l-tryptophanamide, heat-stressed immunoglobulin G (IgG), and bovine serum albumin (BSA) by monitoring changes in steady state fluorescence spectra and time-resolved fluorescence decays as a function of dye concentration. Stronger quenching of the intrinsic BSA and IgG fluorescence in steady state than in time-resolved fluorescence by bis-ANS and ANS pointed toward static quenching being the dominant mechanism in addition to dynamic quenching and/or FRET. In conclusion, one should consider the role of direct excitation of ANS and bis-ANS at 280 nm to ensure a proper interpretation of fluorescence signals resulting from dye-protein interactions. When ANS or bis-ANS is to be used for protein characterization, we recommend selectively exciting the dyes at the higher absorption wavelength maximum (370 or 385 nm, respectively).     

Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid

The structural and functional properties of the nucleocapsid (NC) protein of the avian myeloblastosis virus were examined by steady-state fluorescence and fluorescence anisotropy measurements of the complex between the NC and the extrinsic fluorophore 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid (bis-ANS). The intrinsic fluorescence of bis-ANS is enhanced many fold upon forming a complex with the NC. Between 2 and 10 molecules of bis-ANS bind strongly to the NC, with an overall Kd of less than 10(-6) M. The emission of bis-ANS in the complex can also be induced by excitation at 298 nm, indicating that energy is transferred from Trp 80, the sole tryptophan in the NC protein, to bis-ANS. The energy transferred between the Trp 80 and bis-ANS was analyzed to yield a calculated distance of separation between these fluorophores of 28 +/- 3 A; thus, Trp 80 is well removed from the nearest bound bis-ANS. The fluorescence emission of bis-ANS in the NC.bis-ANS complex is efficiently quenched by added salts and by poly(A), suggesting that salt (presumably anions), nucleic acid, and bis-ANS bind to the same, positively charged region on the NC protein. A site size of six nucleotides was determined for nucleic acid binding to the NC protein, with an estimated Kd of less than 10(-6) M. Salt (anion) binding is strong, but nonspecific, with a Kapp of 4 mM, raising the possibility that anion binding to the NC protein might regulate the interaction of the NC with viral RNA inside the host cell.     

Perturbation of DPPC/POPG bilayers by the N-terminal helix of lung surfactant protein SP-B: a <Superscript>2</Superscript>H NMR study

SP-B_8–25 is a synthetic peptide comprising the N-terminal helix of the essential lung surfactant protein SP-B. Rat lung oxygenation studies have shown that SP-B_8–25 retains some of the function of full-length SP-B. We have used deuterium nuclear magnetic resonance (²H-NMR) to examine the influence of SP-B_8–25 on the mixing properties of saturated PC and unsaturated PG lipids in model mixed lipid bilayers containing dipalmitoylphosphatidylcholine (DPPC) and palmitoyl-oleoyl-phosphatidylglycerol (POPG), in a molar ratio of 7:3. In the absence of the peptide, ²H-NMR spectra of DPPC/POPG mixtures, with one or the other lipid component deuterated, indicate coexistence of large liquid crystal and gel domains over a range of about 10°C through the liquid crystal to gel transition of the bilayer. Addition of SP-B_8–25 has little effect on the width of the transition but the spectra through the transition range cannot be resolved into distinct liquid crystal and gel spectral components suggesting that the peptide interferes with the tendency of the DPPC and POPG lipid components in this mixture to phase separate near the bilayer transition temperature. Quadrupole echo decay observations suggest that the peptide may also reduce differences in the correlation times for local reorientation of the two lipids. These observations suggest that SP-B_8–25 promotes a more thorough mixing of saturated PC and unsaturated PG components and may be relevant to understanding the behaviour of lung surfactant material under conditions of lateral compression which might be expected to enhance the propensity for saturated and unsaturated surfactant lipid components to segregate.     

How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio R<Subscript>Fd</Subscript> of leaves with the PAM fluorometer

This contribution is a practical guide to the measurement of the different chlorophyll (Chl) fluorescence parameters and gives examples of their development under high-irradiance stress. From the Chl fluorescence induction kinetics upon irradiation of dark-adapted leaves, measured with the PAM fluorometer, various Chl fluorescence parameters, ratios, and quenching coefficients can be determined, which provide information on the functionality of the photosystem 2 (PS2) and the photosynthetic apparatus. These are the parameters F_v, F_m, F₀, F_m′, F_v′, NF, and ΔF, the Chl fluorescence ratios F_v/F_m, F_v/F₀, ΔF/F_m′, as well as the photochemical (q_P) and non-photochemical quenching coefficients (q_N, q_CN, and NPQ). q_N consists of three components (q_N = q_E + q_T + q_I), the contribution of which can be determined via Chl fluorescence relaxation kinetics measured in the dark period after the induction kinetics. The above Chl fluorescence parameters and ratios, many of which are measured in the dark-adapted state of leaves, primarily provide information on the functionality of PS2. In fully developed green and dark-green leaves these Chl fluorescence parameters, measured at the upper adaxial leaf side, only reflect the Chl fluorescence of a small portion of the leaf chloroplasts of the green palisade parenchyma cells at the upper outer leaf half. Thus, PAM fluorometer measurements have to be performed at both leaf sides to obtain information on all chloroplasts of the whole leaf. Combined high irradiance (HI) and heat stress, applied at the upper leaf side, strongly reduced the quantum yield of the photochemical energy conversion at the upper leaf half to nearly zero, whereas the Chl fluorescence signals measured at the lower leaf side were not or only little affected. During this HL-stress treatment, q_N, q_CN, and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. q_N was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas q_CN and NPQ decreased with progressive stress even though non-photochemical quenching still continued. It is strongly recommended to determine, in addition to the classical fluorescence parameters, via the PAM fluorometer also the Chl fluorescence decrease ratio R_Fd (F_d/F_s), which, when measured at saturation irradiance is directly correlated to the net CO₂ assimilation rate (_{P N}) of leaves. This R_Fd-ratio can be determined from the Chl fluorescence induction kinetics measured with the PAM fluorometer using continuous saturating light (cSL) during 4–5 min. As the R_Fd-values are fast measurable indicators correlating with the photosynthetic activity of whole leaves, they should always be determined via the PAM fluorometer parallel to the other Chl fluorescence coefficients and ratios.     

Mutational analysis of the hydantoin hydrolysis pathway in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> RU-KM3<Subscript>S</Subscript>

The biocatalytic conversion of 5-mono-substituted hydantoins to the corresponding d-amino acids or l-amino acids involves first the hydrolysis of hydantoin to a N-carbamoylamino acid by an hydantoinase or dihydropyrimidinase, followed by the conversion of the N-carbamoylamino acid to the amino acid by N-carbamylamino acid amidohydrolase (N-carbamoylase). Pseudomonas putida strain RU-KM3_S, with high levels of hydantoin-hydrolysing activity, has been shown to exhibit non-stereoselective hydantoinase and l-selective N-carbamoylase activity. This study focused on identifying the hydantoinase and N-carbamoylase-encoding genes in this strain, using transposon mutagenesis and selection for altered growth phenotypes on minimal medium with hydantoin as a nitrogen source. Insertional inactivation of two genes, dhp and bup, encoding a dihydropyrimidinase and -ureidopropionase, respectively, resulted in loss of hydantoinase and N-carbamoylase activity, indicating that these gene products were responsible for hydantoin hydrolysis in this strain. dhp and bup are linked to an open reading frame encoding a putative transport protein, which probably shares a promoter with bup. Two mutant strains were isolated with increased levels of dihydropyrimidinase but not -ureidopropionase activity. Transposon mutants in which key elements of the nitrogen regulatory pathway were inactivated were unable to utilize hydantoin or uracil as a nitrogen source. However, these mutations had no effect on either the dihydropyrimidinase or -ureidopropionase activity. Disruption of the gene encoding dihydrolipoamide succinyltransferase resulted in a significant reduction in the activity of both enzymes, suggesting a role for carbon catabolite repression in the regulation of hydantoin hydrolysis in P. putida RU-KM3_S cells.     

Characterization of a tryptophan 6-halogenase from <Emphasis Type="Italic">Streptomyces toxytricini</Emphasis>

Tryptophan (Trp) halogenases are found in various bacteria and play an important role in natural product biosynthesis. Analysis of the genome of Streptomyces toxytricini NRRL 15443 revealed an ORF, stth, encoding a putative Trp halogenase within a non-ribosomal peptide synthetase gene cluster. This gene was cloned into pET28a and functionally overexpressed in Escherichia coli. The enzyme halogenated both l- and d-Trp to yield the corresponding 6-chlorinated derivatives. The optimum activity was at 40°C and pH 6 giving k _cat /K _M value of STTH of 72,000 min⁻¹ M⁻¹. The enzyme also used bromide to yield 6-bromo-Trp.     

Hydrolysis of DNA by a Branched Peptide without Aromatic Residues

A branched peptide Nα, Nɛ-di (l-leucyl)-l-lysine was found to efficiently cleave supercoiled double-strand DNA such as PUC19 DNA at optimum pH 4.0 in 40 mmol/l Britton–Robbinson buffer. The T4 ligase experiment implied that the DNA cleavage occurs via a hydrolytic path. The dependence of the cleavage reaction on the ionic strength indicated that the interaction of DNA with the branched peptide involve only electrostatic binding.     

Association of lysozyme with phospholipid vesicles is accompanied by membrane surface dehydration

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between binding of the protein and the subsequent destabilization of the phospholipid vesicles a set of experiments was performed using phospholipid monolayers and vesicles. Using microelectrophoresis the binding of lysozyme to phospholipid vesicles made of PS was determined. At low ionic strength and mild acidic pH of the solution lysozyme reduced the magnitude of the negative zeta potential of PS vesicles at lower concentrations compared to neutral pH and high ionic strength. In contrast, the bound fraction of lysozyme to PS vesicles was nearly constant at acidic and neutral pH. At low pH, the binding of lysozyme was accompanied by a strong aggregation of the vesicles. Lysozyme binding to PS vesicles is accompanied by its penetration into the PL monolayer. This was measured by surface tension and film balance measurements at low pH and low ionic strength. The interaction of lysozyme with negatively charged vesicles lead to a decrease of the vesicle surface hydration as measured by the shift of the emission peak of the fluorescent probe DPE. The binding of bis-ANS increased at low pH after addition of lysozyme to the vesicles. This indicates that more hydrophobic patches of the lysozyme-PS complex are exposed at low pH. At low pH the binding process of lysozyme to PS vesicles was followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the aqueous content of vesicles.     

pH-dependent redox and CO binding properties of chelated protoheme-<Emphasis Type="SmallCaps">l</Emphasis>-histidine and protoheme-glycyl-<Emphasis Type="SmallCaps">l</Emphasis>-histidine complexes

The pH dependence of redox properties, spectroscopic features and CO binding kinetics for the chelated protohemin-6(7)-l-histidine methyl ester (heme-H) and the chelated protohemin-6(7)-glycyl-l-histidine methyl ester (heme-GH) systems has been investigated between pH 2.0 and 12.0. The two heme systems appear to be modulated by four protonating groups, tentatively identified as coordinated H₂O, one of heme’s propionates, Nε of the coordinating imidazole, and the carboxylate of the histidine residue upon hydrolysis of the methyl ester group (in acid medium). The pK _a values are different for the two hemes, thus reflecting structural differences. In particular, the different strain at the Fe–N _ε bond, related to the different length of the coordinating arm, results in a dramatic alteration of the bond strength, which is much smaller in heme-H than in heme-GH. It leads to a variation in the variation of the pK a for the protonation of the N _ε of the axial imidazole as well as in the proton-linked behavior of the other protonating groups, envisaging a cross-talk communication mechanism among different groups of the heme, which can be operative and relevant also in the presence of the protein matrix. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Study of the enhancement of a new chemiluminescence reaction and its application to determination of β‐lactam antibiotics

An enhanced thiosemicarbazide(TSC)–H₂O₂ chemiluminescence (CL) system was established and proposed as a new analytical method for determination of β‐lactam antibiotics, ampicillin sodium and amoxicillin at microgram levels. The method is based on the inhibition of CL emission accompanying oxidation of TSC by H₂O₂ in alkaline medium. The effect of anionic, cationic, and non‐ionic surfactants on the CL emission of the system was studied. Both N‐cetyl‐N,N,N‐trimethylammonium bromide (CTMAB) and Triton X‐100, unlike sodium dodecyl sulfate (SDS), reinforced the CL intensity and were efficient to approximately the same level. The effect of the presence of eight non‐aqueous solvents on the CL system was also investigated. Upon addition of both of the non‐ionic surfactant, Triton X‐100, and the non‐aqueous solvent, N,N‐dimethyl formamide (DMF), the intensity of the CL reaction was increased 100‐fold. This method allows the measurement of 25–545 µg amoxicillin, and 35–350 µg ampicillin sodium. The detection limits are 8 µg for amoxicillin and 9 µg for ampicillin sodium. The relative standard deviations of six replicate measurements of 200 µg amoxicillin and 200 µg ampicillin sodium were 1.9 and 2.1%, respectively. The effect of foreign species on the determination of amoxicillin and ampicillin sodium was also examined. The proposed method was successfully applied to the determination of ampicillin sodium and amoxicillin in some pharmaceutical dosage forms. Copyright © 2008 John Wiley & Sons, Ltd.     

Red-edge-excitation fluorescence spectroscopy of single-tryptophan proteins

With the aim of finding non-equilibrium dipole-relaxational electronic excited states of tryptophan residues in proteins the dependence of the fluorescence emission maximum on excitation wavelength was studied for several proteins containing a single tryptophan residue per molecule. Spectral shifts upon red-edge excitation are not observed for short wavelength-emitting proteins (azurin, two-calcium form of whiting parvalbumin, ribonucleases C ₂ and T ₁). This may be because of the non-polar environment of the tryptophan residues in these proteins or because of the absence of dipole-orientational broadening of spectra. The effect was also not found for proteins emitting at long wavelengths (max. at 341–350 nm) —melittin at low ionic strength, IT-Aj1 protease inhibitor, myelin basic protein. In these proteins, the tryptophan residues are exposed to the rapidly relaxing aqueous solvent. Spectral shifts associated with red-edge excitation are observed for proteins emitting in the medium spectral range — human serum albumin in the N and F forms, IT-Aj1 protease inhibitor at pH 2.9, melittin at high ionic strength as well as the albumin-dodecylsulfate complex. This suggests the existence in these proteins of a distribution of microstates for tryptophan environment with various orientation of dipoles and of slow (on the nanosecond time scale) mobility of the field of these dipoles. As a result the emission proceeds from electronic excited states which are not at equilibrium.     

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3

Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK ₁=0.67±0.16 m,k ₂=1.6±0.7 sec^–1,k _–2=0.17±0.09 sec^–1,K ₁=6.3±1.7 m,k ₂=9±4 sec^–1 andk _–2=7±3 sec^–1. The apparent dissociation constants of chloride from band 3,K _Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.     

Fluorescence and Phosphorescence Study of Tet Repressor–Operator Interaction

Fluorescence and phosphorescence measurements have been carried out on single-p tryptophan (Trp 43 or Trp 75)-containing mutants of Tet repressor (Tet R). Tet R containing Trp 43, the residue localized in the DNA recognition helix of the repressor, has been used to observe the binding of Tet R to two 20-bp DNA sequences of tet O₁ and tet O₂ operators. Binding of Tet R to tet O₁ operator leads to a 78% decrease of the repressor fluorescence intensity, with an accompanying 20-nm blue shift of its fluorescence emission maximum to 330 nm. Upon binding of Tet R to tet O₂ operator, the Trp 43 fluorescence intensity is quenched by 60%, and a 10-nm shift of its emission maximum to 340 nm occurs. Solute fluorescence quenching studies, using acrylamide, performed at low ionic strength indicate that in both the complex of Tet R with the O₁ and that with the O₂ operator, Trp 43 is moderately buried, as indicated by a bimolecular rate quenching constant of about 1.8 × 10⁹ M^–1 sec^–1. In contrast to the Tet R–tet O₂ complex, the Stern–Volmer acrylamide quenching constant K _sv of the complex with tet O₁ operator changes from 7.5 M^–1 at 5 mM NaCl to 22 M^–1 at 200 mM NaCl, indicating different exposures of Trp 43 in the two complexes in solutions of higher ionic strength. Phosphorescence studies showed a 0–0 vibronic transition at 408 and 403 nm for Trp 43 and Trp 75, respectively. Upon binding of Tet R to the tet operators, we observed red shifts of 0–0 vibronic bands of Trp 43 to 413 and 412 nm for tet O₁ and tet O₂ operator, respectively, and the phosphorescence triplet lifetime of Trp 43 at 75 K was quenched from 6.0–5.5 to 3.5–3.3 sec. The thermal phosphorescence quenching profile ranged from –200°C to –20°C, and differed drastically for the two complexes, suggesting different dynamics of the microenvironment of the Trp 43 residue. The luminescence data for Trp 43 of Tet R suggest that the recognition helix of Tet R interacts in different fashions with the tet O₁ and tet O₂ operators.     

Development of an optical sensor for chlortetracycline detection based on the fluorescence quenching of l‐tryptophan

A simple and selective spectrofluorimetric method for the detection of chlortetracycline (CTC) was studied. In pH 7.4 buffer medium l ‐tryptophan (l ‐Trp), applied as the fluorescence probe, interacted with CTC resulting in fluorescence quenching of the probe. CTC was detected with maximum excitation and emission wavelengths at λ_ex/λ_em = 275/350 nm. Notably, quenching of fluorescence intensities was positively proportional to the CTC concentration over the range of 0.65–30 μmol L^?1 and the limit of detection was 0.2 μmol L^?1. Effect of temperature shown in Stern?Volmer plots, absorption spectra and fluorescence lifetime determination, indicated that fluorescence quenching of l ‐Trp by CTC was mainly by static quenching. The proposed study used practical samples analysis satisfactorily.     

C<Subscript>1</Subscript>-/C<Subscript>2</Subscript>-aromatic-imino-glyco-conjugates: experimental and computational studies of binding,inhibition and docking aspects towards glycosidases isolated from soybean and jack bean

Several C₁-imino conjugates of d-galactose, d-lactose and d-ribose, where the nitrogen center was substituted by the salicylidene or naphthylidene, were synthesized and characterized. Similar C₂-imino conjugates of d-glucose have also been synthesized. All the glyco-imino-conjugates, which are transition state analogues, exhibited 100% inhibition of the activity towards glycosidases extracted from soybean and jack bean meal. Among these, a galactosyl-napthyl-imine-conjugate (1c) showed 50% inhibition of the activity of pure α-mannosidase from jack bean at 22 ± 2.5 μM, and a ribosyl-naphthyl-imine-conjugate (3c) showed at 31 ± 5.5 μM and hence these conjugates are potent inhibitors of glycosidases. The kinetic studies suggested non-competitive inhibition by these conjugates. The studies are also suggestive of the involvement of aromatic, imine and carbohydrate moieties of the glyco-imino-conjugates in the effective inhibition. The binding of glyco-imino-conjugate has been established by extensive studies carried out using fluorescence emission and isothermal titration calorimetry. The conformational changes resulted in the enzyme upon interaction of these derivatives has been established by studying the fluorescence quench of the enzyme by KI as well as from the secondary structural changes noticed in CD spectra. All these studies revealed the difference in the binding strengths of the naphthylidene vs. salicylidene as well as galactosyl vs. lactosyl moieties present in these conjugates. The differential inhibition of these glyco-conjugates has been addressed by quantifying the specific interactions present between the glyco-conjugates and the enzyme by using rigid docking studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Excimer fluorescence of pyrene-tropomyosin adducts

Studies of the fluorescence of N-(1-pyrene)maleimide and N-(1-pyrenyl)iodoacetamide with actin from rabbit skeletal muscle tropomyosin revealed the presence of excimer fluorescence characterized by a broad emission band at 480 nm with a shoulder at 505 nm. Monomer fluorescence decay exhibited different lifetimes, viz., about 3, 22 and 87 ns for the pyrenemaleimide adduct; about 2.5, 11 and 51 ns for the aminolyzed maleimide adduct: and about 2.5, 15 and 74 ns for the pyrenyliodoacetamide adduct. Almost identical excimer fluorescence lifetimes were found for all adducts; about 9, 35 and 65 ns. Excimer fluorescence was sensitive to changes in ionic strength and pH of the medium while monomer fluorescence did not change. The protein denaturants guanidine hydrochloride and urea caused dissociation of the two tropomyosin subunits and partial disappearance of excimer fluorescence, but not as effectively as the hydrophobic surfactant sodium dodecyl sulfate. The sensitivity of excimer fluorescence to changes in the micro-environment make these pyrene derivatives very useful probes for studying conformational changes and binding interaction of tropomyosin with other contractile proteins. The unique location of the excimer probe at tropomyosin Cys-190 and its characteristic long lifetimes could make it useful in time-resolved anisotropy studies and fluorescence energy-transfer experiments.     

Comparative studies on the properties of the extrinsic manganese-stabilizing protein from higher plants and of a synthetic peptide of its C-terminus

The present study describes a comparative analysis on the fluorescence properties of the manganese-stabilizing protein (MSP), a synthetic peptide corresponding to its C terminus and a 7:1 (molar ratio) mixture of N-acetyl-tyrosine and N-acetyl-tryptophan, respectively, together with reconstitution experiments of oxygen evolution in MSP-depleted photosystem II (PS II) membrane fragments. It is found: (i) at neutral pH, the fluorescence from Trp(241) is strongly diminished in MSP solutions, whereas it highly dominates the overall emission from the C-terminus peptide; (ii) at alkaline pH, the emission of Tyr and Trp is quenched in both, MSP and C-terminus peptide, with increasing pH but the decline curve is shifted by about two pH units towards the alkaline region in MSP; (iii) a drastically different pattern emerges in the 7:1 mixture where the Trp emission even slightly increases at high pH; (iv) the anisotropy of the fluorescence emission is wavelength-independent (310-395 nm) and indicative of one emitter type (Trp) in the C-terminus peptide and of two emitter types (Tyr, Trp) in MSP; and (v) in MSP-depleted PS II membrane fragments the oxygen evolution is restored (up to 85% of untreated control) by rebinding of MSP but not by the C-terminus peptide, however, the presence of the latter diminishes the restoration effect of MSP. A quenching mechanism of Trp fluorescence by a next neighbored tyrosinate in the peptide chain is proposed and the relevance of the C terminus of MSP briefly discussed.