Protein-lipid interactions and differential scanning calorimetric studies of bacteriorhodopsin reconstituted lipid-water systems

Bacteriorhodopsin has been reconstituted at various molar concentrations into liposomes of dimyristoyl- and also of dipalmitoylphosphatidylcholine. Differential scanning calorimetry indicates that as the protein concentration within the lipid bilayer increases, the cooperativity of the lipid phase transition is reduced, i.e. the transition is broadened, while the midpoint transition temperature remains virtually unchanged. Freeze-fracture electron microscopy of our preparation shows, in agreement with previous data from other laboratories, that extensive protein aggregation occurs when the liposome is cooled below the T_c transition temperature of the lipid. Laser flash photolysis measurements of protein rotation of the bacteriorhodopsin show, especially in the case of protein-rich recombinants, that protein aggregates exist even above T_c. The perturbation caused by the presence of bacteriorhodopsin in the lipid bilayer is similar to that produced by other intrinsic proteins. The difficulty of correlating the observed calorimetric enthalpy data with a simple concept of a ‘boundary lipid layer’ based upon consideration of a single isolated protein is discussed in view of the occurrence of protein aggregates both above and below T_c. It is concluded that the reduction of enthalpy is related to the number of lipids which solvate the protein aggregates within the protein-lipid patches and are thereby removed from the cooperative melting and enthalpy of the remaining regions of pure lipid.     

A high-sensitivity differential scanning calorimetric study of the interaction of melittin with dipalmitoylphosphatidylcholine fused unilamellar vesicles

High-sensitivity differential scanning calorimetry has been used to examine the interaction of bee venom melittin with dipalmitoylphosphatidylcholine fused unilamellar vesicles. Experiments were performed under conditions for which melittin in solution is either monomeric (in low salt) or tetrameric (in high salt). It was found that under both sets of conditions melittin abolishes the pretransition at a relatively high lipid-to-protein molar incubation ratio, Ri (about 200) and that at intermediate values of Ri it broadens the main transition profile and reduces the transition enthalpy. This provides evidence which suggests that melittin is at least partially inserted into the apolar region of the bilayer. Evident at low values of Ri are two peaks in the lipid thermal transition profiles, which may arise from a heterogeneous population of lipid vesicles formed through fusion induced by melittin, or by lipid phase separation. For those profiles which exhibited only one peak, transition enthalpies, normalized to those of the lipid in the absence of the protein, are plotted vs. the bound protein-to-lipid molar ratios for the experiments performed under the conditions which give monomeric and tetrameric melittin in solution. These plots yield straight lines, the slopes of which give the number of lipid molecules each protein molecule excludes from participating in the phase transition. These were found to be 9.9 +/- 0.7 and 4.1 +/- 0.5 for monomeric and tetrameric melittin, respectively. The results are discussed in terms of possible models for the binding of melittin to phospholipid vesicles. For simple hexagonal packing of lipid molecules, incorporation as an aggregate is favored when melittin is tetrameric in solution, whereas incorporation as a monomer is favored when melittin is monomeric in solution. For low-salt solutions, evidence is obtained for the contribution of free melittin to lipid fusion, perhaps by the formation of protein bridges between apposed vesicles.     

Differential scanning calorimetric studies of ethanol interactions with distearoylphosphatidylcholine: transition to the interdigitated phase

It is well established that ethanol and other amphipathic molecules induce the formation of a fully interdigitated gel phase in saturated like-chain phosphatidylcholines (PC's). We have previously shown that the induction of interdigitation in PC's by ethanol is dependent upon the alcohol concentration, the lipid chain length, and the temperature [Nambi, P., Rowe, E. S. & McIntosh, T. J. (1988) Biochemistry 27, 9175-9182]. In the present study, we have used high-sensitivity differential scanning calorimetry to investigate the transitions of distearoylphosphatidylcholine between the noninterdigitated and the interdigitated phases. The enthalpy of the L beta' to L beta I transition is approximately half that of the L beta' to P beta' transition which occurs in the absence of ethanol. The reversibility of these transitions has also been investigated by employing both heating and cooling scans in order to establish the most stable phases as a function of temperature and ethanol concentration. It has been demonstrated that the transition to the interdigitated phase is reversible as a function of temperature. Kinetic studies on the reverse transition (L beta I to L beta') demonstrate that this transition can be very slow, requiring weeks to reach completion. The rate depends upon temperature and ethanol concentration. The slow phase changes mean that the lipid can exist for long periods of time in a phase structure which is not the most stable state. The biological significance of this type of lipid behavior is the implication that the phase structure of biological membranes may depend not only on the most stable phase structure of the lipids present but also on the synthetic pathway or other kinetic variables.     

A differential scanning calorimetric study of brain clathrin

The thermal denaturation of clathrin-coated vesicles isolated from bovine brain tissue has been studied by differential scanning calorimetry and has been compared to basket structures reformed from isolated triskelion trimers of clathrin and to isolated triskelions. The coated vesicles and reformed baskets displayed similar, yet distinct, thermal behavior. Calorimetric data of the coated vesicles exhibited a single denaturation transition peak at 55.9 +/- 0.1 degrees C, skewed to low temperatures whereas the thermograms for the reformed baskets exhibited a broad transition peak at 53.1 +/- 0.1 degrees C and a peak at 56.3 +/- 0.1 degrees C. Neither transition was reversible. The specific transition enthalpy was 11.5 +/- 1.0 J g-1 for the coated vesicles and the total transition enthalpy was 9.1 +/- 0.3 J g-1 for the reformed baskets. In contrast, isolated triskelions showed no thermal transition between 15 and 90 degrees C. Although the coated vesicles and the reformed baskets have similar stability reflecting their similar structures, the coated vesicles appear to be marginally more stable than the reformed baskets. The complexity of the transition profiles and their lack of symmetry suggest the existence of several, somewhat independent, domains unique to the cage-like structure of the coated vesicles and reformed baskets.     

Structure of Colorado potato beetle lipophorin: differential scanning calorimetric and small-angle X-ray scattering studies

The structure of lipophorin, isolated from hemolymph of the Colorado potato beetle, was investigated by differential scanning calorimetry (DSC) and small-angle X-ray scattering. The DSC heating curves of intact lipophorin showed endothermic peaks that were similar to peaks obtained with the hydrocarbon fraction isolated from this lipophorin. The observed peaks correlated with the transition of the hydrocarbons from an ordered into a more disordered state. Changes in structure of the lipophorin particles with increasing temperature were also observed by small-angle X-ray scattering studies. The structural organization of lipophorin was further elucidated by simulation analysis, using a three-layered symmetrical sphere as a model. These studies revealed that lipophorin from the Colorado potato beetle is a sphere with a maximum diameter of 175 A. The sphere is composed of three radially symmetrical layers of different electron densities. The outer layer (37.5-39.5 A in thickness) is composed of phospholipid, apolipophorin I, and part of apolipophorin II. The middle layer (5-10 A) contains diacylglycerol, the rest of apolipophorin II, and probably beta-carotene. The core of the particle (40-45 A) only contains hydrocarbons. This structure differs from another model, previously proposed for cockroach and locust lipophorins [Katagiri, C., Sato, M., & Tanaka N. (1987) J. Biol. Chem. 262, 15857-15861], in the small size of the middle layer. The volume of the middle layer correlated well with the low diacylglycerol content of this lipophorin.     

Thermal denaturation of a recombinant mouse amelogenin: circular dichroism and differential scanning calorimetric studies

Conformational analyses of a recombinant mouse tooth enamel amelogenin (rM179) were performed using circular dichroism (CD), fluorescence, differential scanning calorimetry, and sedimentation equilibrium studies. The results show that the far-UV CD spectra of rM179 at acidic pH and 10 degrees C are different from the spectra of random coil in 6 M GdnHCl. A near-UV CD spectrum of rM179 at 10 degrees C is similar to that of rM179 in 6 M GdnHCl, which indicates that aromatic residues of native structure are exposed to solvent and rotate freely. Far-UV CD values of rM179 at 80 degrees C are different from that of random-coil structure in 6 M GdnHCl, which suggests that rM179 at 80 degrees C has specific secondary structures. A gradual thermal transition was observed by far-UV CD, which is interpreted as a weak cooperative transition from specific secondary structures to other specific secondary structures. The fluorescence emission maximum for the spectrum due to Trp residues in rM179 at 10 degrees C shows the same fluorescence emission maximum as rM179 in 6 M GdnHCl and amino acid Trp, which indicates that the three Trp in rM179 are exposed to solvent. Deconvolution of differential scanning calorimetry curve gives the population of three states (A, I, and C states). These results indicate that three states (A, I, and C) have specific secondary structures, in which hydrophobic and Trp residues are exposed to the solvent. The thermodynamic characteristics of rM179 are unique and different from a typical globular protein, proline-rich peptides, and a molten globule state.     

A differential scanning calorimetric study of the bovine lens crystallins

Differential scanning calorimetry was performed on the five major lens crystallin fractions [HM-alpha, alpha, beta H, beta L, and (beta s + gamma)] of the bovine lens as well as on more purified forms of alpha- and gamma-crystallins. All were found to be relatively thermally stable although the alpha-crystallin were found to at least partially unfold at an approximately 10 degrees C lower temperature than the beta and gamma fractions. Increasing protein concentration had little effect on gamma-crystallin thermograms but had marked effects on those of the alpha- and beta-crystallins. Increases in the thermal stability with increasing protein concentration for the beta-crystallins can be explained most simply by the known beta L/beta H equilibrium, but, in the case of the alpha-crystallins, excluded volume effects may be an important factor. In both cases, the increased stability at high concentrations could be of physiological relevance. As well as the expected endothermic unfolding transitions, all of the lens crystallins revealed exothermic peaks that correlate with protein precipitation. Interestingly, this phenomenon occurs only after extensive structural alteration in the case of the alpha-crystallins but is present very early in the initial stages of structural perturbation of the beta- and gamma-crystallins.     

Differential scanning calorimetric study of the effect of sterol side chain length and structure on dipalmitoylphosphatidylcholine thermotropic phase behavior.

We have investigated the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers containing a series of cholesterol analogues varying in the length and structure of their alkyl side chains. We find that upon the incorporation of up to approximately 25 mol % of any of the side chain analogues, the DPPC main transition endotherm consists of superimposed sharp and broad components representing the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. Moreover, the behavior of these components is dependent on sterol side chain length. Specifically, for all sterol/DPPC mixtures, the sharp component enthalpy decreases linearly to zero by 25 mol % sterol while the cooperativity is only moderately reduced from that observed in the pure phospholipid. In addition, the sharp component transition temperature decreases for all sterol/DPPC mixtures; however, the magnitude of the decrease is dependent on the sterol side chain length. With respect to the broad component, the enthalpy initially increases to a maximum around 25 mol % sterol, thereafter decreasing toward zero by 50 mol % sterol with the exception of the sterols with very short alkyl side chains. Both the transition temperature and cooperativity of the broad component clearly exhibit alkyl chain length-dependent effects, with both the transition temperature and cooperativity decreasing more dramatically for sterols with progressively shorter side chains. We ascribe the chain length-dependent effects on transition temperature and cooperativity to the hydrophobic mismatch between the sterol and the host DPPC bilayer (see McMullen, T. P. W., Lewis, R. N. A. H., and McElhaney, R. N. (1993) Biochemistry 32:516-522).(ABSTRACT TRUNCATED AT 250 WORDS)     

Polymorphism of N-stearoylethanolamine: differential scanning calorimetric,vibrational spectroscopic (FTIR), and crystallographic studies

Based on a series of physicochemical properties (differential scanning calorimetry, powder X-ray crystallographic studies and Fourier-transform infra red spectroscopic analysis) determined for N-stearoylethanolamine (NSEA) (C18:0) at different temperatures, evidence has been given that this compound can exist in (at least) three polymorphic forms. Powder X-ray crystallography clearly demonstrates the presence of three distinct molecular packings at distinct temperatures while spectral changes in the vibrational spectra reveal that the geometry of the CH(2)z.sbnd;CO functional group of the molecule is affected during the polymorphic transitions. Rationalization of the thermal physicochemical behavior of NSEA in terms of molecular packing is also proposed. It supposes rearrangement of the hydrocarbon chains upon heating of the molecule.     

Stabilization of actin by phalloidin: a differential scanning calorimetric study.

We have used differential scanning calorimetry to study the effects of phalloidin on F and G actin stability. For F actin, saturating concentrations of phalloidin induced an important shift on the transition temperature, Tm, from 69.5 degrees C to 83.5 degrees C. However, the calorimetric enthalpy remained unchanged. Using lower phalloidin concentrations, monomers linked to phalloidin, as well as neighboring unlinked monomers, were both stabilized. Contrary to previous reports, phalloidin was also shown to affect G actin, shifting its Tm from 59.5 degrees C to 75 degrees C. Two mechanisms are proposed to explain this finding: first, it could indicate a real interaction of phalloidin with G actin, and second, heating of the specimen during the scan could have induced polymerization of some G actin to the F form. The resulting F polymer would then interact with phalloidin, thus shifting the equilibrium between G and F actin towards the polymeric form.     

Pressure perturbation and differential scanning calorimetric studies of bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

Differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) were used to characterize thermal phase transitions, membrane packing, and volumetric properties in multilamellar vesicles (MLVs) composed of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at different temperatures. For PLFE MLVs derived from cells grown at 78 degrees C, the first DSC heating scan exhibits an endothermic transition at 46.7 degrees C, a small hump near 60 degrees C, and a broad exothermic transition at 78.5 degrees C, whereas the PPC scan reveals two transitions at approximately 45 degrees C and 60 degrees C. The endothermic peak at 46.7 degrees C is attributed to a lamellar-to-lamellar phase transition and has an unusually low DeltaH (3.5 kJ/mol) and DeltaV/V (0.1%) value, as compared to those for the main phase transitions of saturated diacyl monopolar diester lipids. This result may arise from the restricted trans-gauche conformational changes in the dibiphytanyl chain due to the presence of cyclopentane rings and branched methyl groups and due to the spanning of the lipid molecules over the whole membrane. The exothermic peak at 78.5 degrees C probably corresponds to a lamellar-to-cubic phase transition and exhibits a large and negative DeltaH value (-23.2 kJ/mol), which is uncommon for normal lamellar-to-cubic phospholipid phase transformations. This exothermic transition disappears in the subsequent heating scans and thus may involve a metastable phase, which is irreversible at the scan rate used. Further, there is no distinct peak in the plot of the thermal expansion coefficient alpha versus temperature near 78.5 degrees C, indicating that this lamellar-to-cubic phase transition is not accompanied by any significant volume change. For PLFE MLVs derived from cells grown at 65 degrees C, similar DSC and PPC profiles and thermal history responses were obtained. However, the lower growth temperature yields a higher DeltaV/V ( approximately 0.25%) and DeltaH (14 kJ/mol) value for the lamellar-to-lamellar phase transition measured at the same pH (2.1). A lower growth temperature also generates a less negative temperature dependence of alpha. The changes in DeltaV/V, DeltaH, and the temperature dependence of alpha can be attributed to the decrease in the number of cyclopentane rings in PLFE at the lower growth temperature. The relatively low DeltaV/V and small DeltaH involved in the phase transitions help to explain why PLFE liposomes are remarkably thermally stable and also echo the proposal that PLFE liposomes are generally rigid and tightly packed. These results help us to understand why, despite the occurrence of thermal-induced phase transitions, PLFE liposomes exhibit a remarkably low temperature sensitivity of proton permeation and dye leakage.     

Time-resolved x-ray diffraction and calorimetric studies at low scan rates: I. Fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases

The phase transitions in fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases have been studied by lowangle time-resolved x-ray diffraction under conditions similar to those employed in calorimetry (scan rates 0.05-0.5°C/min and uniform temperature throughout the samples). This approach provides more adequate characterization of the equilibrium transition pathways and allows for close correlations between structural and thermodynamic data. No coexistence of the rippled gel (P_β') and liquid-crystalline (L_α) phases was found in the main transition of DPPC; rather, a loss of correlation in the lamellar structure, observed as broadening of the lamellar reflections, takes place in a narrow temperature range of ~100 mK at the transition midpoint. Formation of a long-living metastable phase, denoted by P_β'(mst), differing from the initial P_β' was observed in cooling direction by both x-ray diffraction and calorimetry. No direct conversion of P_β'(mst) into P_β' occurs for over 24 h but only by way of the phase sequence P_β'(mst) → L_β' → P_β'. According to differential scanning calorimetry (DSC), the enthalpy of the P_β'(mst)-L_α transition is by ~5% lower than that of the P_β'-L_α transition. The effects of ethanol (Rowe, E. S. 1983. Biochemistry. 22:3299-3305; Simon, S. A., and T. J. McIntosh. 1984. Biochim. Biophys. Acta 773:169-172) on the mechanism and reversibility of the DPPC main transition were clearly visualized. At ethanol concentrations inducing formation of interdigitated gel phase, the main transition proceeds through a coexistence of the initial and final phases over a finite temperature range. During the subtransition in DPPC recorded at scan rate 0.3°C/min, a smooth monotonic increase of the lamellar spacing from its subgel (L_c) to its gel (L_β') phase value takes place. The width of the lamellar reflections remains unchanged during this transformation. This provides grounds to propose a “sequential” relaxation mechanism for the subgel-gel transition which is not accompanied by growth of domains of the final phase within the initial one.     

Theoretical basis for differential scanning calorimetric analysis of multimeric proteins

A new general equation simulating irreversible DSC transitions of multimeric proteins was developed. The equation put forward here is the result of an improved mathematical re-elaboration of the classical Lumry-Eyring models, where no restrictive a priori assumptions are made on the kinetic constraints of the denaturation process, or on the enthalpy of the final denatured state. In order to test the wide applicability of this new effective theoretical tool, a series of DSC transitions were simulated with the aim of determining the effects of all relevant thermodynamic, kinetic or experimental parameters on the shape of DSC profiles. Moreover, the classical equations used widely in DSC investigations for the calculus in both kinetic parameters and changes of molecularity, were studied in the light of the model developed here, highlighting, in each case, their rather limited applicability. The new approach proposed in this article was applied to study the thermal denaturation of an hexameric protein (Glucosamine-6-phosphate deaminase), putting in evidence the practical applicability of the theoretical equations developed.     

A differential scanning calorimetric study of the thermal unfolding of apo- and holo-cytochrome b562.

Cytochrome b562 is a four-helix-bundle protein containing a non-covalently bound b-type heme prosthetic group. In the absence of heme, cytochrome b562 remains highly structured under native conditions. Here we report thermodynamic data for the thermal denaturation of the holo- and apoproteins as determined by differential scanning calorimetry. Thermal denaturation of holocytochrome b562 is a highly reversible process, and unexpectedly does not involve dissociation of the heme prosthetic group. Thermal denaturation of the corresponding apoprotein, with the heme group chemically removed, remains a cooperative, reversible process. Apocytochrome b562 is substantially destabilized relative to the holoprotein: the t1/2 is more than ten degrees lower, and enthalpy and heat capacity changes are about one-half of the holoprotein values. However, the energetic parameters of apocytochrome b562 denaturation are within the range of observed values for small proteins.     

Differential scanning calorimetric and theoretical studies of ColE1 DNA

The differential scanning calorimetry (DSC) of plasmid ColE1 DNA was carried out. The DSC curve under the solvent condition of 1.0 X SSC buffer gave eleven clear peaks over the temperature range of 83 to 98 degrees C. The DSC curves obtained here were essentially in good agreement with the optical melting curves of ColE1 DNA reported previously. The theoretical melting profiles of ColE1 DNA calculated from its entire nucleotide sequence showed a good agreement with the DSC curves. The theoretical analysis made by constructing the thermal stability map showed that there was the positional correlation between the boundaries of the cooperatively melting regions and the ends of the protein coding regions of genes of ColE1. It was shown that the helix-coil transition of many of the small genes had a single cooperatively melting region. However, the large genes such as cea and mob3 had two or more cooperatively melting regions. It was suggested that this is closely related to the domain structures of the proteins encoded by such genes.     

Differential scanning calorimetric studies of a Bacillus halodurans alpha-amylase

The thermal unfolding of Amy 34, a recombinant alpha-amylase from Bacillus halodurans, has been investigated using differential scanning calorimetry (DSC). The denaturation of Amy 34 involves irreversible processes with an apparent denaturation temperature (T(m)) of 70.8 degrees C at pH 9.0, with four transitions, as determined using multiple Gaussian curves. The T(m) increased by 5 degrees C in the presence of 100-fold molar excess of CaCl2 while the aggregation of Amy 34 was observed in the presence of 1000-fold molar excess of CaCl2. Increase in the calcium ion concentration from 1- to 5-fold molar excess resulted in an increase in calorimetric enthalpy (DeltaH(cal)), however, at higher concentrations of CaCl2 (up to 100-fold), DeltaH(cal) was found to decrease, accompanied by a decrease in entropy change (DeltaS), while the T(m) steadily increased. The presence of 100-fold excess of metal chelator, EDTA, resulted in a decrease in T(m) by 10.4 degrees C. T(m) was also decreased to 61.1 degrees C and 65.9 degrees C at pH 6.0 and pH 11.0, respectively.     

A differential scanning calorimetric study of chymotrypsin in the presence of added polymers

Scanning calorimetry measurements of different amounts of chymotrypsin in water alone gave a temperature of denaturation (T(d)) value of 54 degrees C. However, when high-molecular-weight poly(ethylene glycol) was added to aqueous solutions of chymotrypsin, the thermostability of the enzyme was enhanced. For example, the addition of 20% (w/w) of poly(ethylene glycol) of molecular weight of 100,000 increased the T(d/) value to 66 degrees C. In toluene containing various amounts of added water, ethyl cellulose was used to improve the thermostability of chymotrypsin. For this system, a T(d) value of 82 degrees C was obtained with a 20% (w/w/) concentration of ethyl cellulose and 2% (v/v) of added water. Polymers in these solvents interact with water, which could otherwise denature the enzyme; polymers also from complexes with enzyme molecules to produce a more stable structure. (c) 1994 John Wiley & Sons, Inc.