Chang-Hwei Chen

Comparison of the stability of phycocyanins from thermophilic, mesophilic, psychrophilic and halophilic algae

Protein unfolding of eight different phycocyanins was investigated utilizing circular dichroism and visible spectra. The phycocyanin samples were extracted from algae that are normally found in vastly different environments, and are classified as mesophilic, thermophilic, halophilic and psychrophilic. The ability of these proteins to resist the denaturant urea is in the order of thermophile greater than mesophile, halophile greater than psychrophile. Based on a two-state approximation the apparent free energies of protein unfolding at zero urea denaturant concentration, deltaGH2Oapp, were found to range from 2.4 to 8.8 kcal/mole for the eight phycocyanins at pH 6 and 25 degrees C. The proteins from the thermophile are generally more stable than those from the mesophile. An extra stability of the halophile is believed due to the specific interaction of the proteins and the ions in solution. A correction for deltaGH2Oapp due to minor amino acid differences reveals that the stability and the structural properties of these proteins are primarily affected by this minor difference in amino acid compositions.

Denaturatin of phycocyanin by urea and determination of the enthalpy of denaturation by microcalorimetry

Denaturation of the protein phycocyanin in urea solution was investigated by microcalorimetry, ultraviolet and visible spectroscopy, circular dichroism and sedimentation equilibrium. The results consistently demonstrated that in the presence of 7 M urea this protein is completely denatured. By assumings a two-state mechanism, an apparent free energy of unfolding at zero denaturant concentration, (formula: see text) was found to be 4.4 kcal/mole at pH 6.0 and 25 degrees C. By microcalorimetry the enthalpy of denaturation of phycocyanin app was found to be -230 kcal/mole at 25 degrees C. The relatively large negative enthalpy change results from protein unfolding and changes in protein solvation.

PHOTOSENSITIVITY OF ARTIFICIAL BILAYER MEMBRANES: LIPID‐CHLOROPHYLL INTERACTION

Abstract— The interaction between lipid and chlorophyll in the photosensitive model bilayer membranes was studied by investigating the effects of the hydrocarbon chain length (6–20 carbons), degree of saturation (0–3 double bonds), and head group character of a series of synthetic and natural lipids on the membrane photoresponse. The results suggest, as a general phenomenon, that any stable membrane formed in the lipid‐chlorophyll a‐β‐carotene system is photosensitive regardless of the type of lipid in the membrane. The magnitude of the photoresponse (ΔV) varies for different lipids, and membranes containing natural phospholipids have a higher ΔV. The presence of chlorophyll a in a lipid solution enhances the lipid membrane stability.

Thermodynamic elucidation of solute-induced lipid interdigitation phase: Lipid interactions with hydrophobic versus amphipathic species

Comparative thermodynamic studies on the interactions of aqueous dispersions of dipalmitoyl phosphatidylcholine (DPPC) bilayer vesicles with hydrophobic and amphipathic species were conducted to elucidate the nature of the solute-induced interdigitated lipid phase. Cyclohexanol, a strong hydrophobic species, lowers the temperature (tm) of the lipid main phase transition from the gel to the liquid-crystalline phase. Unlike ethanol (an amphipathic species), as reported previously, cyclohexanol does not exert a biphasic effect on tm (lowering tm at lower concentrations and raising tm at higher concentrations). At cyclohexanol greater than or equal to 15.4 mg/ml or 0.154 M, the thermogram of DPPC vesicles exhibits a small transition adjacent to the main phase transition but at a lower temperature. In contrast, ethanol does not promote such a small transition. Furthermore, the enthalpy (delta H) of the transition is increased in the presence of cyclohexanol. The sign of the enthalpy change (delta H-delta Ho) is positive and that of the free energy change (delta G-delta Go) is negative, a characteristic of solute-solute hydrophobic interaction. In contrast, DPPC bilayer vesicles exhibit both (delta H-delta Ho) and (delta G-delta Go) greater than 0 in the presence of ethanol in a concentration range where lipid vesicles exist in an interdigitated phase. To support the above distinct thermodynamic observations, fluorescence steady-state polarization (P) measurements were also performed. At the temperature below tm, the value of P decreases as cyclohexanol concentration increases, while a biphasic effect on P was found in the presence of ethanol. These findings support the postulation that the solute-induced interdigitated lipid phase requires the solute molecule to be amphipathic in nature.

Comparative thermodynamic elucidation of the structural stability of thermophilic proteins.

Differential scanning calorimetry, circular dichroism, and visible absorption spectrophotometry were employed to elucidate the structural stability of thermophilic phycocyanin derived from Cyanidium caldarium, a eucaryotic organism which contains a nucleus, grown in acidic conditions (pH 3.4) at 54 degrees C. The obtained results were compared with those previously reported for thermophilic phycocyanin derived from Synechococcus lividus, a procaryote containing no organized nucleus, grown in alkaline conditions (pH 8.5) at 52 degrees C. The temperature of thermal unfolding (t(d)) was found to be comparable between C. caldarium (73 degrees C) and S. lividus (74 degrees C) phycocyanins. The apparent free energy of unfolding (DeltaG([urea]=0)) at zero denaturant (urea) concentration was also comparable: 9.1 and 8.7 kcal/mole for unfolding the chromophore part of the protein, and 5.0 and 4.3 kcal/mole for unfolding the apoprotein part of the protein, respectively. These values of t(d) and DeltaG([urea]=0) were significantly higher than those previously reported for mesophilic Phormidium luridum phycocyanin (grown at 25 degrees C). These findings revealed that relatively higher values of t(d) and DeltaG([urea]=0) were characteristics of thermophilic proteins. In contrast, the enthalpies of completed unfolding (DeltaH(d)) and the half-completed unfolding (DeltaH(d)) 1 2 for C. caldarium phycocyanin were much lower than those for S. lividus protein (89 versus 180 kcal/mole and 62 versus 115 kcal/mole, respectively). Factors contributing to a lower DeltaH(d) in C. caldarium protein and the role of charged groups in enhancing the stability of thermophilic proteins were discussed.

Energetic studies of lactose active transport in Escherichia coli membrane vesicles

The energetics of D-lactate-driven active transport of lactose in right-side-out Escherichia coli membrane vesicles has been investigated with a microcalorimetric method. Changes of enthalpy (delta Hox), free energy (delta Gox), and entropy (delta Sox) during the D-lactate oxidation reaction in the presence of membrane vesicles are -39.9 kcal, -46.4 kcal, and 22 cal/deg per mole of D-lactate, respectively. The free energy released by this reaction is utilized to form a proton electrochemical potential (delta-microH+) across the membrane. The higher observed heat in the D-lactate oxidation reaction in the presence of carbonylcyanide m-chlorophenylhydrazone (a proton ionophore) supports the postulate that delta-microH+ is formed across the membrane vesicles. Thermodynamic quantities for the formation of delta-microH+ are delta Hm = 14.1 kcal, delta Gm = 0.6 kcal, and delta Sm = 45 cal/deg per mole of D-lactate. The efficiency in the free energy transfer from the oxidation reaction to the formation of delta-microH+ (defined by delta Gm/delta Gox) was 2%, as compared to that in the heat transfer (defined by delta Hm/delta Hox) of 35%. The energetics of the movement of lactose in symport with proton across the membrane as a consequence of the formation of delta-microH+ are delta H1 = -19 kcal, delta G1 = -0.5 kcal, and delta S1 = -62 cal/deg per mole of lactose. No heat of reaction is contributed by lactose movement across the membrane without symport with H+.

Association and dissociation of phycocyanin by small molecules

Abstract Sedimentation velocity experiments showed that tetraalkylammonium salts, with alkyl chain lengths ranging from methyl to pentyl and in the concentration range from 0.02 to 0.16 m , decrease the aggregation of C-phycocyanin in sodium phosphate buffers, pH 6.0 and 7.0, I 0.1, at 23 °C. Tetrabutylammonium and tetrapentylammonium bromides and chlorides disaggregate 11S and 17S C-phycocyanin aggregates to produce a 6S subunit. The larger alkyl group produces the greater effect. An explanation for this effect would be that hydrophobic and possibly ionic interactions between the protein and the tetraalkylammonium cation effectively block assembly of the 6S to 11S aggregate. These salts may provide a novel means for the control of protein assembly. Recent studies on the effect of neutral aromatic compounds on C-phycocyanin aggregation were extended to saturated solutions of α- and β-naphthol, with concentrations of m , respectively. Sedimentation velocity experiments and absorption spectroscopy suggest that the aggregation of C-phycocyanin is increased by naphthol binding to the protein.

Comparison of the energetics of lactose active transport: Artificial versus enzyme-associated energy source☆

To further consider the thermochemical method as a useful approach for active transport research and to investigate the characteristic of a proton electrochemical potential (delta mu H+) across the membrane, the energetics of lactose active transport across Escherichia coli membrane vesicles coupled with an artificial electron donor (phenazine methosulfate-ascorbate) has been investigated. The results were compared with those obtained with an enzyme-associated electron donor (lactate dehydrogenase-D-lactate). The oxidation of an electron donor provided the energy necessary for the transport process. The observed higher heat of ascorbate oxidation reaction in the presence of a proton ionophore (carbonyl cyanide m-chlorophenylhydrazone) further confirmed the formation of delta mu H+ across the membrane. Part of the oxidation energy was utilized to form delta mu H+. Comparison of the energetics revealed that the magnitudes of delta Hox (the enthalpy of the oxidation reaction) and delta Hm (the enthalpy of the formation of delta mu H+) in the two energy sources were comparable (-46 kcal/mol of ascorbate to -40 kcal/mol of D-lactate for delta Hox and 9.6 kcal/mol of ascorbate to 14 kcal/mol of D-lactate for delta Hm). Comparable and low value (about 1%) was also found in the free energy transfer (defined by delta Gm/delta Gox) from the oxidation reaction to the formation of delta mu H+. These results, in combination with the close values of delta mu H+ observed in the two systems, suggested that the characteristic of the created delta mu H+ was independent of the energy source. Examination of delta Hm might provide the information on the ratio of the number of protons produced, as 1 mol of two different electron donors was oxidized. The oxidation reaction in the presence of membrane vesicles was discussed.

The Modification by Biliproteins of Intensity and Direction of Electron Flow Across Chlorophyll-Containing Membranes

Photosensitive phenomena in artificial bileaflet membranes containing photosynthetic pigments are described and analyzed in terms of a potential model for the primary events in photo-synthetic energy storage. Illumination of a bilayer membrane interposed between aqueous solutions of different redox potential results in establishment of an electric current across the membrane. It is suggested that absorption of photons by the chlorophyll in the bilayer allows electron transfer across the membrane. This process differs from photosynthetic processes in two important aspects: the quantum efficiency of the process in the synthetic membrane is very low and energy is dissipated rather than stored. When biliproteins (extrinsic membrane proteins from blue-green and red algae) are introduced into the system, the resulting membranes exhibit two important properties: an increase by two- to threefold of the quantum efficiency of the electron transfer process and an abilty to direct the flow of electrons even against a pre-existing potential gradient, so that energy storage is accomplished. The observations are analyzed in terms of a model to explain the processes relevant to photosynthesis and the trivial process of degradation of the redox gradient.