Soumen Basak

Caspase 8 mediated apoptotic cell death induced by β-sheet forming polyalanine peptides

Expansion of a polyalanine stretch from 10 to 12–17 residues in the N‐terminus of the protein PABP2 has been implicated in the genetically acquired disease oculopharyngeal muscular dystrophy, characterized by nuclear protein deposits. Here we report a correlation between the structural properties and cell toxicity of two peptides mimicking the N‐terminal domain of PABP2: one containing seven and the other 11 uninterrupted alanine residues. Consistent with earlier observations, the longer peptide (11‐ala) was found to adopt β‐sheet structure while the shorter one (7‐ala) formed α‐helix over a wide range of concentrations (∼20–500 μM). We observed that treatment with 11‐ala resulted in significantly enhanced death of Chinese hamster V79 cells, compared to the effect of treatment with 7‐ala, via the cytochrome c mediated apoptotic pathway. Increases in caspase 8 and caspase 3 activity were also observed in human cells (K562) treated with 11‐ala. These results indicate that the toxicity of pathogenic peptides is directly linked to their β‐sheet structure and also support recent observations that small oligomeric species of peptides and proteins are the key toxic elements in causing protein aggregation diseases.

Burst dynamics during drainage displacements in porous media: Simulations and experiments

We investigate the burst dynamics during drainage going from low to high injection rate at various fluid viscosities. The bursts are identified as pressure drops in the pressure signal across the system. We find that the statistical distribution of pressure drops scales according to other systems exhibiting self-organized criticality. The pressure signal was calculated by a network model that properly simulates drainage displacements. We compare our results with corresponding experiments.

Conformational kinetics of triligated hemoglobin

We have used the method of modulated excitation (Ferrone, F.A., and J.J. Hopfield, 1976, Proc. Natl. Acad. Sci. USA. 73:4497-4501), with an improved apparatus and a revised analytical procedure, to measure the rate of conformational change between the oxy (R) and deoxy (T) conformations of triligated carboxy-hemoglobin A at pH 6.5 and 7.0. We have found the rates to be kRT = 1.2 X 10(3) s-1 and kTR = 3.5 X 10(3) s-1 for pH 6.5, while for pH 7.0, kRT = 1.0 X 10(3) s-1, and kTR = 3.0 X 10(3) s-1. The value for L3, the equilibrium constant between conformations, was virtually unchanged between pH 6.5 and 7.0. While the rates measured here differ from those obtained in the original use of this method, these new rates are fully consistent with the original data when analyzed by the revised procedures presented here. When taken with other kinetic and equilibrium data, our measurements suggest that the transition state between structures is dominated by the behavior of the T quaternary structure. Finally, a spectral feature near the HbCO Soret peak has been observed that we ascribe to an allosteric perturbation of the spectra of the liganded hemes.

Interaction with Al and Zn induces structure formation and aggregation in natively unfolded caseins.

Caseins are phosphoproteins that form the principal protein component of milk, their chief function being the transport of inorganic calcium and phosphate to the neonates. The four major members of the casein family are alpha(s1)-, alpha(s2)- (together referred to as alpha(s)-casein), beta- and kappa-casein, each having a characteristic high negative net charge as well as high hydrophobicity and preferring extended conformational states in solution. We have investigated the influence of the polyvalent metal cations Zn(II) and Al(III) on the structure of bovine caseins, using fluorescence and circular dichroic (CD) spectroscopy and light scattering. Changes in Trp and ANS fluorescence parameters (blue shifts of the emission maxima and enhancement of fluorescence intensity) and in the far-UV CD spectra of the caseins caused by the presence of both metals suggest that conformational changes are induced in them by low concentrations (20-40 microM) of the metal cations. These changes lead to formation of solvent-accessible hydrophobic clusters or cavities that, in turn, cause self-association and precipitation of caseins at higher concentration of the metals. These conclusions are supported by increased binding of ThT to the caseins, as well as enhancement of light scattering intensity, observed in presence of Al(III). The chaperonic property of alpha(s)-casein, which enables it to inhibit thermal aggregation of alcohol dehydrogenase, is shown to be partially destroyed by Zn(II)-induced structural alterations, due possibly to loss of flexibility of the natively unfolded casein chains.

Hetero-aggregation with sucrase affects the activity, stability and conformation of extra- and intra-cellular cellobiase in the filamentous fungus T. clypeatus

Cellobiase (C) from T. clypeatus was found to be aggregated with sucrase (S) in extra- and intra-cellular fractions, when co-aggregates of the enzyme with sucrase with different activity ratios (C/S) were obtained during purification. The co-aggregates were compared for their activity, stability, and kinetic parameters with a purified sucrase-free cellobiase preparation. The specific activity and stability of both the extra- and intra-cellular enzyme decreased significantly in the absence of sucrase. The catalytic activity (Vmax/Km) of sucrase-free cellobiase were decreased by 4236 and 652 fold compared to the crude enzyme in culture filtrate and mycelial extracts respectively. The stability of the enzyme also decreased versus pH, temperature and in the presence of chaotropic agents such as SDS, Gdn.HCl and urea after disaggregation from sucrase. Optimum temperatures of the free intra- and extra-cellular cellobiase were shifted to 47°C from 45°C after the removal of sucrase from the co-aggregates, whereas optimum pH of the free enzyme and co-aggregates remained the same. Intra-cellular cellobiase had very high affinity for sucrase and it was difficult to separate them. Cellobiase preparations from extra- and intra-cellular fractions were analysed by circular dichroism and light scattering spectroscopy and it was concluded that co-aggregation with sucrase was responsible for a change in conformation of cellobiase in the aggregates. The conformation of intra-cellular enzyme preparations were also different from those in the extra-cellular fractions. Instant regain of cellobiase activity in intra- and extra-cellular preparations were obtained on the addition in vitro of free sucrase from the respective fractions to the incubation mixture. The experiments suggested that hetero-aggregation with sucrase regulates the activity and stability of cellobiase in the fungus.

SOMATOSTATIN IN A WATER-RESTRICTED ENVIRONMENT: FLUORESCENCE AND CIRCULAR DICHROISM STUDY IN AOT REVERSE MICELLES

Abstract—

Fluorescence study of melanocyte stimulating hormones in AOT reverse micelles.

Fluorescence emission from the single tryptophan residues of two melanocyte stimulating hormones, alpha-MSH and delta-MSH, and their quenching kinetics were studied in aqueous solution and in reverse micelles of AOT/water/isooctane. Incorporation into micelles caused blue shifted and narrower emission peaks, altered quantum yields and considerably enhanced anisotropies for both peptides when compared to emission from bulk water. The variation of emission parameters with micellar water content was interpreted to suggest that while the tryptophan in alpha-MSH lies in close vicinity of the water-AOT molecular interface, that in delta-MSH is solubilized in the central water pool. Total emission intensity decays followed complex (biexponential) kinetics in both aqueous and micellar media. Although the mean lifetimes for both peptides were always nearly the same, the average rotational correlation times in micelles for alpha-MSH were three times as much as those for delta-MSH. Stern-Volmer plots obtained using acrylamide and CCl4 as quenchers localized in the micellar and organic pseudophases, respectively, were non-linear and dependent on emission wavelength. Quenching by acrylamide was more efficient for delta-MSH than for alpha-MSH, while the opposite was true for quenching by CCl4. The implication of this result for localization of the peptides in micelles was consistent with the earlier one emerging from these studies.

Regulation (Alteration) of Activity and Conformation of Sucrase by Coaggregation with Cellobiase in Culture Medium of Termitomyces clypeatus

Extracellular sucrase (S) of Termitomyces clypeatus was aggregated with cellobiase (C) in culture filtrate and coaggregates of sucrase to cellobiase with different activity ratios (S/C) were obtained during purification. Specific activity of the enzyme decreased significantly, after purification of sucrase free from cellobiase. Purified sucrase was characterized as a glycoprotein of molar mass around 55kDa as indicated by SDS–PAGE and HPGPLC. Km and Vmax of the purified enzyme were determined as 34.48 mM and 13.3 U/mg, respectively, at optimum temperature (45 °C) and pH (5.0). Substrate affinity and reaction velocity of the purified enzyme, free from cellobiase, was lowered by approximately 3.5 and 55 times, respectively, than that of the enzyme obtained from culture filtrate. The instant regain of sucrase activity up to the extent of 41% was obtained on in vitro addition of cellobiase (free from sucrase) to the enzyme in incubation mixture. Conformation of the enzyme free from cellobiase appeared to be significantly different from that of the coaggregate, as analyzed by circular dichroic and light scattering spectroscopy. It was concluded that activity and conformation of sucrase is regulated (altered) by heteroaggregation with cellobiase in the fungus.

Sensing of hydrophobic cavity of serum albumin by an adenosine analogue: fluorescence correlation and ensemble spectroscopic studies.

Adenosine is a naturally occurring purine nucleoside that plays important role in various biochemical processes. We have studied the binding of TNP-Ado (trinitrophenylated-adenosine), a fluorescent analogue of adenosine (which itself is a weak fluorophore), with a model transport protein, bovine serum albumin (BSA). The binding affinity was determined using Fluorescence correlation spectroscopy (FCS) and compared with its value obtained from macroscopic fluorescence spectroscopic studies. Fluorescence and circular dichroism (CD) spectroscopies were employed together with molecular docking study to locate the probable binding site of TNP-Ado on BSA and its effect on the conformation and stability of BSA. Fluorescence studies showed that TNP-Ado binds to BSA in 1:1 stoichiometry via an entropically favoured process. Induced CD spectra revealed that a chiro-optical switching of TNP-Ado occurs upon binding to BSA. Results on urea-induced denaturation of BSA and docking study suggested that the binding site for the ligand is in the hydrophobic subdomain IIA of BSA, consistent with the results of other measurements. This study establishes TNP-Ado as a sensor of hydrophobic regions in proteins like serum albumin, having the capability of detecting a minimum concentration of 140ng/ml protein. FCS measurement of binding interaction of rhodamine-labeled TNP-Ado (RTNP-Ado) with BSA yielded an association constant of KFCS=(1.03±0.06) × 10(4)M(-1). The association constants (Ka) obtained for binding of BSA with rhodamine-free (i.e. TNP-Ado) and rhodamine-labeled (RTNP-Ado) ligands, obtained using the ensemble spectroscopic technique, were (2.3±0.06) × 10(5)M(-1) and (3.4±0.03) × 10(4)M(-1), respectively. The difference between the values of Ka for the free and labeled ligands suggests that fluorescent labeling of small molecules perceptibly interferes with the binding process. On the other hand, the difference in Ka obtained by FCS and ensemble techniques is due to the fact that while the former measures the change in the diffusion constant (i.e. size) of RTNP-Ado upon binding to BSA, the latter focuses on the change of tryptophan emission properties of BSA due to the presence of bound RTNP-Ado.

Effect of cholesterol on interaction of dibucaine with phospholipid vesicles: a fluorescence study.

Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoyl phosphatidylcholine (DOPC) containing different mol percents of cholesterol has been studied by fluorescence spectroscopy. Fluorescence measurements on dibucaine in presence of phospholipid vesicles containing various amounts of cholesterol yielded a pattern of variation of wavelength at emission maximum and steady-state anisotropy which indicated that the microenvironment of dibucaine is more polar and flexible in membranes that contain cholesterol than in membranes without cholesterol. Experiments on quenching of fluorescence from membrane-associated dibucaine by potassium iodide showed a marked increase in quenching efficiency as the cholesterol content of the vesicles was increased, demonstrating increased accessibility of the iodide quenchers to dibucaine in the presence of cholesterol, when compared to that in its absence. Total emission intensity decay profiles of dibucaine yielded two lifetime components of approximately 1 ns and approximately 2.8--3.1 ns with mean relative contributions of approximately 25 and approximately 75%, respectively. The mean lifetime in vesicles was 20--30% smaller than in the aqueous medium and showed a moderate variation with cholesterol content. Fluorescence measurements at two different temperatures in DMPC SUVs, one at 33 degrees C, above the phase transition temperature and another at 25 degrees C, around the main phase transition, indicated two different mode of dibucaine localization. At 25 degrees C dibucaine partitioned differentially in presence and absence of cholesterol. However, at 33 degrees C the apparent partition coefficients remained unaltered indicating differences in the microenvironment of dibucaine in presence and absence of cholesterol in the phospholipid membranes.