Alkali-induced conformational transition in different domains of bovine serum albumin

Alkaline pH induced conformational changes in different domains of bovine serum albumin were studied by using domain specific ligands: chloroform, bilirubin and diazepam for domains I, II and III respectively. The effect of alkaline pH on the secondary structure of BSA was monitored by far-UV CD in the range 250 nm to 200 nm. The pH profiles of BSA in the alkaline region showed a two-step change, one corresponding to N<-->B transition (pH 7.5 to 9.0) and the other to B --> U (pH 11.0 to 13.5). Binding of chloroform decreased continuously on increasing pH, whereas binding of diazepam, remained unchanged up to pH 9 and decreased thereafter. In contrast, binding of bilirubin gradually increased up to pH 11.0 and decreased thereafter reaching a value similar to one obtained with native BSA at pH 11.5. Above pH 11.5, bilirubin binding decreased and was abolished completely at pH 12.5. In the pH region 7.5 to 11.0, a continuous decrease in chloroform binding (pH 7.5 to 9.5) and a late decrease in diazepam binding (pH 9.5 to 11.0) suggested major loss of native conformation of domain I followed by domain III during alkaline induced unfolding of BSA. However, a significant increase in bilirubin binding showed a favorable conformational rearrangement in domain II in this pH region (pH7.5 to 11.0). Further, a nearly complete abolishment of bilirubin binding to BSA and significant loss of secondary structure around pH 12.5 indicated that domain II was more resistant to alkaline pH and unfolds only at extreme alkalinity. Taken together, these data suggest that unfolding of three domains of BSA follow the following order of susceptibility towards alkaline denaturation of BSA domain I>domain III>domain II.     

Characterization of histamine H1 binding sites on human T lymphocytes by means of 125I-iodobolpyramine. Preferential expression of H1 receptors on CD8 T lymphocytes

The presence of histamine H1 receptors on lymphocytes has been indirectly suggested by the various effects of agonists or antagonists on the functionally distinct T lymphocyte subsets. Recently, a new H1 antagonist, 125I-iodobolpyramine, whose structure is similar to mepyramine, has become available for the detection of H1 receptors in guinea pig brain. When using 125I-iodobolpyramine on human T lymphocytes, the presence of a single highly specific H1 binding site was evidenced. The binding of 125I-iodobolpyramine to human T cells was reversible when using 1000-fold excess of the cold H1 antagonist, d-chlorpheniramine. Binding saturation was achieved at 0.60-0.65 nM of 125I-iodobolpyramine, the binding equilibrium was reached in 20-30 min at 27 degrees C. The dissociation constant was KD = 0.41 +/- 0.07 (mean +/- SE) and the number of receptors per T cell was 3407 +/- 592 (mean +/- SE) as deduced from saturation and kinetic curves. In competition experiments using a panel of H1 ligands, the T cell binding sites detected by 125I-iodobolpyramine showed a pharmacological behavior characteristic of histamine H1 receptors. It was of particular interest that 125I-iodobolpyramine binding displayed clearcut stereoselectivity as assessed by the higher affinity of the d-configuration of chlorpheniramine than the l form. Study of purified CD4 and CD8 T cells showed that twice as much H1 histamine receptors were expressed by CD8 T lymphocytes (6615 +/- 1125) as compared to CD4 T cells (3545 +/- 459). These results underline the need for studying the functional properties of such pharmacologically defined T lymphocyte H1 binding sites.     

Interaction of diiodo-L-tyrosine and triiodophenol with bovine serum albumin. Circular dichroism and fluorescence studies.

As a model study to investigate the binding mechanism between thyroid hormones and carrier protein, the interaction of diiodo-L-tyrosine (DIT) and triiodophenol (I3phi) with bovine serum albumin (BSA) was investigated by circular dichroism (CD) and fluorescence methods. In both the DIT-BSA system and the I3phi-BSA system, induced Cotton effect was observed in the wavelength region near 320 nm. This induced Cotton effect was measured at various molar ratios of ligands to BSA (L/P). The value of the ellipticity at 319 nm, [theta]319, in the I3phi-BSA system was remarkably large compared with that of the DIT-BSA system, and [theta]319 at an L/P ratio of one was -1.96 X 10(4) (degree cm2 decimole-1) for the I3phi-BSA system and -0.1 X 10(4) for the DIT-BSA system. The binding constants for the combination of BSA with a single molecule of ligand, calculated by measuring the quenching of the fluorescence of the protein, were 1.33 X 10(5) M(-1) at 15 degrees for the DIT-BSA system and 1.6 X 10(9) M(-1) at 28 degrees for the I3theta-BSA system. These results suggest that the binding of I3theta to BSA is stronger than that of DIT and a cleft may exist more congruent with the molecular dimensions of I3theta than with those of DIT.     

Spectroscopic study of the interaction of gossypol with bovine serum albumin

The interaction of gossypol with bovine serum albumin (BSA) at pH 7.6 in 0.02 M borax-borate buffer has been followed by circular dichroism (CD) and difference spectroscopy. From the extrinsic CD band at 390 nm, a binding constant of 2.7 X 10(3) M-1 was calculated. At 54 degrees the induced CD spectrum was abolished, suggesting that the interaction is not favoured at that temperature. The effect of various solvents and salts on the interaction has been followed by difference spectroscopy. The modification of epsilon-amino groups of lysine did not affect the interaction. Binding of gossypol to BSA does not cause a change in its secondary structure or sedimentation coefficient.     

Multiple In Vitro and In Vivo Regulatory Effects of Budesonide in CD4+ T Lymphocyte Subpopulations of Allergic Asthmatics

Background

Increased activation and increased survival of T lymphocytes characterise bronchial asthma.

Objectives

In this study the effect of budesonide on T cell survival, on inducible co-stimulator T cells (ICOS), on Foxp3 and on IL-10 molecules in T lymphocyte sub-populations was assessed.

Methods

Cell survival (by annexin V binding) and ICOS in total lymphocytes, in CD4+/CD25+ and in CD4+/CD25- and Foxp3 and IL-10 in CD4+/CD25+ and in CD4+/CD25-cells was evaluated, by cytofluorimetric analysis, in mild intermittent asthmatics (n = 19) and in controls (n = 15). Allergen induced T lymphocyte proliferation and the in vivo effects of budesonide in mild persistent asthmatics (n = 6) were also explored.

Results

Foxp3 was reduced in CD4+/CD25- and in CD4+/CD25+ cells and ICOS was reduced in CD4+/CD25+ cells but it was increased in CD4+CD25-in asthmatics when compared to controls. In asthmatics, in vitro, budesonide was able to: 1) increase annexin V binding and to reduce ICOS in total lymphocytes; 2) increase annexin V binding and Foxp3 and to reduce ICOS in CD4+/CD25- cells; 3) reduce annexin V binding and to increase IL-10 and ICOS in CD4+/CD25+ cells; 4) reduce cell allergen induced proliferation. In vivo, budesonide increased ICOS in CD4+/CD25+ while it increased Foxp3 and IL-10 in CD4+/CD25+ and in CD4+/CD25- cells.

Conclusions

Budesonide modulates T cell survival, ICOS, Foxp3 and IL-10 molecules differently in T lymphocyte sub-populations. The findings provided shed light on new mechanisms by which corticosteroids, drugs widely used for the clinical management of bronchial asthma, control T lymphocyte activation.     

Equilibrium study on the binding between thermolysin and Streptomyces metalloprotease inhibitor, talopeptin (MKI)

The binding between thermolysin and its specific inhibitor, talopeptin (MKI), was found to show a fluorescence increase when excited at 280 nm and 295 nm, and a difference spectrum characterized by two peaks at 294 nm and 285 nm with a shoulder around 278 nm, indicating a microenvironmental change in tryptophan residue(s) of thermolysin and/or talopeptin. The inhibitor constant of talopeptin against thermolysin, Ki, was determined over the pH range 5-9 from the inhibition of the enzyme activity towards 3-(2-furylacryloyl)-glycyl-L-leucine amide (FAGLA) as a substrate. The dissociation constant of thermolysin-talopeptin complex, Kd, determined directly from fluorometric titration was in good agreement with the inhibitor constant, Ki, between pH 6 and 8.5. The pH dependence of Ki and Kd suggested that at least two ionizable groups of thermolysin in their protonated forms are essential for the binding between thermolysin and talopeptin. The temperature dependence of K1 at pH 5.5 indicated that the binding is largely exothermic (delta H degree = -12 kcal/mol) and essentially enthalpy-driven.     

Binding of Lipoic Acid Induces Conformational Change and Appearance of a New Binding Site in Methylglyoxal Modified Serum Albumin

The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.     

Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling

The binding of several different categories of small molecules to bovine (BSA) and human (HSA) serum albumins has been studied for many years through different spectroscopic techniques to elucidate details of the protein structure and binding mechanism. In this work we present the results of the study of the interactions of BSA and HSA with the anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) monitored by fluorescence spectroscopy of the intrinsic tryptophans at pH 5.0. Similarly to pH 7.0 and 9.0, at low concentrations, the interaction of BSA with these surfactants shows a quenching of fluorescence with Stern-Volmer quenching constants of (1.1+/-0.1)x10(4) M(-1), (3.2+/-0.1)x10(3) M(-1) and (2.1+/-0.1)x10(3) M(-1) for SDS, HPS and CTAC, respectively, which are associated to the 'effective' association constants to the protein. On the interaction of these surfactants with HSA, an opposite effect was observed as compared to BSA, i.e., an enhancement of fluorescence takes place. For both proteins, at low surfactant concentrations, a positive cooperativity was observed and the Hill plot model was used to estimate the number of surfactant binding sites, as well as the association constants of the surfactants to the proteins. It is worthy of notice that the binding constants for the surfactants at pH 5.0 are lower as compared to pH 7.0 and 9.0. This is probably due to fact that the protein at this acid pH is quite compact reducing the accessibility of the surfactants to the hydrophobic cavities in the binding sites. The interaction of myristic acid with both proteins shows a similar fluorescence behaviour, suggesting that the mechanism of the interaction is the same. Recently published crystallographic studies of HSA-myristate complex were used to perform a modelling study with the aim to explain the fluorescence results. The crystallographic structure reveals that a total of five myristic acid molecules are asymmetrically bound in the macromolecule. Three of these sites correspond to higher affinity ones and correlate with high association constants described in the literature. Our models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid. The differences in tryptophan vicinity upon surfactant binding are explored in the models in order to explain the observed spectroscopic changes. For BSA the quenching is due to a direct contact of a surfactant molecule with the indole of W131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W131, corresponds to a lower affinity site, explaining the lower binding constants obtained from fluorescence studies. In the case of HSA the enhancement of fluorescence is due to the removal of static quenching of W214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan.     

Binding characteristics of a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine, to bovine serum albumin as measured by fluorescence

The interaction between a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine and bovine serum albumin was investigated by fluorescence measurements. The apparent binding constants were obtained at various pHs assuming the equivalence and independence of the interaction sites on the protein from the fluorescence titration curves. The maximum binding was attained at pH 8.0, and the apparent binding constant was (5.28 +/- 0.13).10(5) M-1 with one binding site per albumin molecule. Thermodynamic parameters were also determined from the van't Hoff plot of the apparent binding constants at pH 7.5. The free energy change, enthalpy change and entropy change were -7.70 +/- 0.09 kcal.mol-1, -4.59 kcal.mol-1 and 10.2 e.u., respectively.     

Structural and thermodynamic studies of KM+, a d-mannose binding lectin from Artocarpus integrifolia seeds

The KM+ lectin exhibits a novel and unusual circular dichroism (CD) spectrum that could be explained by a high proline content that would be inducing deformation of the beta-structure and/or unusual turns. KM+ was shown to be a very rigid lectin, which was very stable under a broad variety of conditions (urea, guanidine, hydrolysis, pH, etc.). Only incubation for 60 min at 333-338 K and extreme basic pH were able to induce conformational changes which could be observed by CD and fluorescence measurements. Data from CD are typical for protein denaturing associated with changes in the overall secondary structure. Data from high-performance size exclusion chromatography (SEC) showed that the denatured forms produced at pH 12.0 are eluted in clusters that co-elute with the native forms. A significant contribution from the tyrosines to the fluorescence emission upon denaturation was observed above 328 K. In fact at 328 K some broadening of the emission spectrum takes place followed by the appearance of a shoulder (approx. 305 nm) at 333 K and above. The sensitivity of tryptophan fluorescence to the addition of sugar suggests a close proximity of the tryptophan residues to the sugar binding site, K(a)=(2.9+/-0.6)x10(3) M(-1). The fraction of chromophore accessible to the quencher obtained is f(a)=0.43+/-0.08, suggesting that approximately 50% of the tryptophan residues are not accessible to quenching by d-mannose. KM+ thermal denaturation was found to be irreversible and was analyzed using a two-state model (N-->D). The results obtained for the activation energy and transition temperature from the equilibrium CD studies were: activation energy, E(a)=134+/-11 kJ/mol and transition temperature, T(m)=339+/-1 K, and from the fluorescence data: E(a)=179+/-18 kJ/mol and T(m)=337+/-1 K. Kinetic studies gave the following values: E(a)=108+/-18 kJ/mol and E(a)=167+/-12 kJ/mol for CD and fluorescence data, respectively.     

Examination of structural characteristics of the potent oxytocin antagonists [dPen1,Pen6]-OT and [dPen1,Pen6, 5-tBuPro7]-OT by NMR, Raman, CD spectroscopy and molecular modeling.

The synthesis and biological evaluation of penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs and comparison with their proline(7)-oxytocin counterparts has led to the discovery of two potent oxytocin (OT) antagonists: [dPen(1),Pen(6)]-oxytocin (1, pA(2) = 8.22, EC(50) = 6.0 nM) and [dPen(1),Pen(6),5-tBuPro(7)]-oxytocin (2, pA(2) = 8.19, EC(50) = 6.5 nM). In an attempt to understand the conformational requirements for their biological activity, spectroscopic analyses of 1 and 2 were performed using (1)H NMR, laser Raman and CD techniques. In H(2)O, oxytocin analogs 1 and 2 exhibited cis-isomer populations of 7% and 35%, respectively. Measurement of the amide proton temperature coefficients revealed solvent shielded hydrogens for Gln(4) and Pen(6) in the major trans-conformer of 1 as well as for Gln(4) in the minor cis-conformer of 2. Few long-distance NOEs were observed, suggesting conformational averaging for analogs 1 and 2 in water; moreover, a lower barrier (16.6 +/- 0.2 kcal/mol) for isomerization of the amide N-terminal to 5-tBuPro(7) relative to OT was calculated from measuring the coalescence temperature of the Gly(9) backbone NH signals in the NMR spectra of 2. Observed bands in the Raman spectra of 1 and 2 correspond to C(beta)-S-S-C(beta) dihedral angles of +110-115 degrees and +/-90 degrees , respectively. In water, acetonitrile and methanol, the CD spectra for 1 exhibited a positive maximum around 236-239 nm; in trifluoroethanol, the spectra shifted and a negative maximum was observed at 240 nm. The CD spectra of 2 were unaffected by solvent changes and exhibited a negative maximum at 236-239 nm. The CD and Raman data both suggested that a conformation having a right-handed screw sense about the disulfide and a chi(CS-SC) dihedral angle value close to 115 degrees was favored for analog 1 in water, methanol and acetonitrile, but not trifluoroethanol, where a +/-90 degrees angle was favored. Analog 2 was more resilient to conformational change about the disulfide, and adopted a preferred disulfide geometry corresponding to a +/-90 degrees chi(CS-SC) dihedral angle. Monte Carlo conformational analysis of analogs 1 and 2 using distance restraints derived from NMR spectroscopy revealed two prominent conformational minima for analog 1 with disulfide geometries around +114 degrees and +116 degrees . Similar analysis of analog 2 revealed one conformational minimum with a disulfide geometry around +104 degrees . In sum, the conformation about the disulfide in [dPen(1),Pen(6)]-OT (1) was shown to be contingent on environment and in TFE, adopted a geometry similar to that of [dPen(1),Pen(6),5-tBuPro(7)]-OT (2) which appeared to be stabilized by hydrophobic interactions between the 5-tBuPro(7) (5R)-tert-butyl group, the Leu(8) isopropyl sidechain and the Pen(6)beta-methyl substituents. In light of the conformational rigidity of 2 about the disulfide bond, and the similar geometry adopted by 1 in TFE, a S-S dihedral angle close to +110 degrees may be a prerequisite for their binding at the receptor.     

Characterization of the interaction between reserpine and bovine serum albumin: spectroscopic approaches

The characteristics of the interaction between reserpine and bovine serum albumin (BSA) were studied by fluorescence, UV-vis absorption and Fourier transform infrared (FT-IR) spectroscopy. Spectroscopic analysis revealed that fluorescence quenching of BSA by reserpine was through a static quenching procedure. The binding constant K(A) of reserpine with BSA at 293, 301 and 309 K was 1.63, 1.78 and 2.35 x 10(5) moL(-1) L respectively, which indicated degree of binding force between reserpine and BSA. There was one binding site between reserpine and BSA. The entropy and enthalpy changes were positive, indicating that interaction of reserpine and BSA was driven mainly by hydrophobic forces. The average binding distance between the donor (BSA) and the acceptor (reserpine) was about 3.84 nm based on the Forster non-radiation energy transfer theory. Results of synchronous fluorescence and FT-IR spectra indicated that the conformation and microenvironment of BSA were changed by the binding of reserpine. The results may provide important insights into the physiological activity of reserpine.     

Kinetics and mechanism of bilirubin binding to human serum albumin

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.     

A ‘turn‐on’ fluorescent chemosensor for quantification of serum albumin in aqueous solution at neutral pH

A fluorescent chemosensor 1 (4‐diethylamino‐2′‐hydroxychalcone) for detecting serum albumin with long‐wavelength emission, good selectivity and facile synthesis was reported. Upon the addition of bovine serum albumin (BSA) to an aqueous solution of 1 at neutral pH, a ‘turn‐on’ fluorescence response was observed at 596 nm based on a hydrophobic binding mode between 1 and BSA. A linear range of 0.10–1.00 mg/mL and a detection limit of 9.1 µg/mL for BSA were obtained, respectively. Moreover, 1 was successfully applied to detect BSA in real bovine serum samples with satisfied recovery and accuracy, which suggested that 1 could serve as a valid and effective fluorescent chemosensor for quantification of BSA. Copyright © 2015 John Wiley & Sons, Ltd.     

Equilibrium and transient intermediates in folding of human macrophage migration inhibitory factor.

Acid, guanidinium-Cl and urea denaturations of recombinant human macrophage migration inhibitory factor (MIF) were measured using CD and fluorimetry. The acid-induced denaturation was followed by CD at 200, 222, and 278 nm and by tryptophan fluorescence. All four probes revealed an acid-denatured state below pH 3 which resembled a typical molten globule. The pH transition is not two-state as the CD data at 222 nm deviated from all other probes. Urea and guanidinium-Cl denaturations (pH 7, 25 degrees C) both gave an apparent DeltaGU app H2O of 31 +/- 3 kJ.mol-1 when extrapolated to zero denaturant concentration. However, denaturation transitions recorded by fluorescence (at the same protein concentration) occurred at lower urea or guanidinium-Cl concentrations, consistent with an intermediate in the course of MIF denaturation. CD at 222 nm was not very sensitive to protein concentration (in 10-fold range) even though size-exclusion chromatogryphy (SEC) revealed a dimer-monomer dissociation prior to MIF unfolding. Refolding experiments were performed starting from acid, guanidinium-Cl and urea-denatured states. The kinetics were multiphasic with at least two folding intermediates. The intrinsic rate constant of the main folding phase was 5.0 +/- 0.5 s-1 (36.6 degrees C, pH 7) and its energy of activation 155 +/- 12 kJ.mol-1.     

Lymphocyte subset analyses in healthy adults vaccinated with yellow fever 17DD virus

In this study the kinetics of humoral and cellular immune responses in first-time vaccinees and re-vaccinees with the yellow fever 17DD vaccine virus was analyzed. Flow cytometric analyses were used to determine percentual values of T and B cells in parallel to the yellow fever neutralizing antibody production. All lymphocyte subsets analyzed were augmented around the 30th post vaccination day, both for first-time vaccinees and re-vaccinees. CD3+ T cells increased from 30.8% (SE +/- 4%) to 61.15% (SE +/- 4.2%), CD4+ T cells from 22.4% (SE +/- 3.6%) to 39.17% (SE +/- 2%) with 43% of these cells corresponding to CD4+CD45RO+ T cells, CD8+ T cells from 15.2% (SE +/- 2.9%) to 27% (SE +/- 3%) with 70% corresponding to CD8+CD45RO+ T cells in first-time vaccinees. In re-vaccinees, the CD3+ T cells increased from 50.7% (SE +/- 3%) to 80% (SE +/- 2.3%), CD4+ T cells from 24.9% (SE +/- 1.4%) to 40% (SE +/- 3%) presenting a percentage of 95% CD4+CD45RO+ T cells, CD8+ T cells from 19.7% (SE +/- 1.8%) to 25% (SE +/- 2%). Among CD8+CD38+ T cells there could be observed an increase from 15 to 41.6% in first-time vaccinees and 20.7 to 62.6% in re-vaccinees. Regarding neutralizing antibodies, the re-vaccinees presented high titers even before re-vaccination. The levels of neutralizing antibodies of first-time vaccinees were similar to those presented by re-vaccinees at day 30 after vaccination, indicating the success of primary vaccination. Our data provide a basis for further studies on immunological behavior of the YF 17DD vaccine.     

Structural analysis of peptides that interact with Newcastle disease virus

A peptide with the sequence CTLTTKLYC has previously been identified to inhibit the propagation of Newcastle disease virus (NDV) in embryonated chicken eggs and tissue culture. NDV has been classified into two main groups: the velogenic group, and mesogenic with lentogenic strains as the other group based on its dissociation constants. In this study the peptide, CTLTTKLYC, displayed on the pIII protein of a filamentous M13 phage was synthesized and mutated in order to identify the amino acid residues involved in the interactions with NDV. Mutations of C1 and K6 to A1 and A6 did not affect the binding significantly, but substitution of Y8 with A8 dramatically reduced the interaction. This suggests that Y8 plays an important role in the peptide-virus interaction. The three-dimensional structure of the peptide was determined using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular modeling. The peptide exhibited two possible conformers. One that consists of consecutive beta-turns around T2-L3-T4-T5 and K6-L7-Y8-C9. The other conformer exhibited a beta-hairpin bend type of structure with a bend around L3-T4-T5-K6.     

Acid-induced equilibrium folding intermediate of human platelet profilin

The acid-induced unfolding of human platelet profilin (HPP) can be minimally modeled as a three-state process. Equilibrium unfolding studies have been performed on human platelet profilin1 (HPP) and monitored by far-UV circular dichroism, tryptophan fluorescence, ANS binding, and NMR spectroscopy. Far-UV CD measurements obtained by acid titration demonstrate that HPP unfolds via a three-state mechanism (N --> I --> U), with a highly populated intermediate between pH 4 and 5. Approximately 80% of native helical secondary structural content remains at pH 4, as indicated by monitoring the CD signal at 222 nm. The stability (DeltaGH2O) of the native conformation at pH 7.0 (obtained by monitoring the change in tryptophan signal as a function of urea concentration) is 5.56 +/- 0.51 kcal mol-1; however, the DeltaGH2O for the intermediate species at pH 4 is 2.01 +/- 0.47 kcal mol-1. The calculated m-values for the pH 7.0 and pH 4.0 species were 1.64 +/- 0.15 and 1.34 +/- 0.17 kcal mol-1 M-1, respectively, which is an indication that the native and intermediate species are similarly compact. Additionally, translational diffusion measurements obtained by NMR spectroscopy and ANS binding studies are consistent with a globular and compact conformation at both pH 7.0 and 4.0. The pKa values for the two histidine (His) residues located on helix 4 of HPP were determined to be 5.6 and 5.7 pH units. These pKa values coincide with the midpoint of the far-UV CD acid titration curve and suggest that the protonation of one or both His residues may play a role in the formation of the unfolding intermediate. Stable intermediate species populate the 2D 1H-15N HSQC NMR spectra between pH 4 and 5. A number of backbone and side-chain resonances show significant perturbations relative to the native spectrum; however, considerable nativelike tertiary contacts remain. Interestingly, the residues on HPP that are significantly altered at low pH coincide with segments of the G-actin binding surface and poly-l-proline binding interface. The earlier reports that a decrease in pH below 6.0 induces structural alterations in profilin, favoring dissociation of the profilin-actin complex, corresponds with the structural alterations observed in the partially unfolded species. Our findings suggest that a novel mechanism for pH induced disruption of the profilin-G-actin complex involve a nativelike unfolding intermediate of profilin.     

Effect of low pH on single skeletal muscle myosin mechanics and kinetics

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity (V(actin)) in the IVM by 36%. Single molecule experiments, at 1 microM ATP, decreased the average unitary step size of myosin (d) from 10 +/- 2 nm (pH 7.4) to 2 +/- 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 microM to 1 mM at pH 6.4 restored d (9 +/- 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. V(actin), however, was not restored by raising the [ATP] (1-10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding (t(on)). Measurement of t(on) as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs t(on) by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., V(actin) approximately d/t(on)), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in V(actin), suggesting a molecular explanation for this component of muscular fatigue.     

Manganese, calcium, and saccharide binding to concanavalin A, as studied by ultrafiltration

The binding of the ligands Mn2+, Ca2+, and methyl alpha-D-glucopyranoside to concanavalin A, purified as described (A.J. Sophianopoulos and J.A. Sophianopoulos (1981) Prep. Biochem. 11, 413-435), was studied by ultrafiltration in 0.2 M NaCl, pH 5.2 and pH 6.5 to 7, and at 23 to 25 degrees C. The association constant (Ka) of methyl alpha-D-glucopyranoside to concanavalin A was (2 +/- 0.2) X 10(3) M-1, both at pH 5.2 and 7. At pH 5.2 and in the absence of Ca2+, the Ka of Mn2+ to concanavalin A was (5 +/- 1) X 10(3) M-1, and in the presence of 1 mM Ca2+, the Ka was (9.1 +/- 2.1) X 10(5) M-1. At pH 6.5 Mn2+ bound to concanavalin A with a Ka of (7.3 +/- 1.8) X 10(5) M-1, and the binding affinity was virtually independent of the presence of Ca2+. Experiments of binding of 4-methylumbelliferyl alpha-D-mannopyranoside to concanavalin A indicated that at pH 5.2, binding of a single Mn2+ per concanavalin A monomer was sufficient to induce a fully active saccharide binding site. Ca2+ is not necessary for such activation, but rather it increases the affinity of concanavalin A for binding Mn2+.