Fourier transform infrared spectroscopy suggests unfolding of loop structures precedes complete unfolding of pig citrate synthase

Pig citrate synthase (PCS) can be used as a model enzyme to gain some insight into the structural basis of protein thermostability. The thermal unfolding characteristics of the specific secondary structure elements within PCS were monitored in detail by following changes in its amide I band components. The result of our study indicates that PCS undergoes irreversible thermal denaturation. Detailed analysis reveals that the different secondary structures display a multistep transition with a major and a minor transition at different temperatures and a very small initial transition at the same temperature (30 degrees C). A plot of temperature-induced changes in (1)H-(2)H exchange, the decrease in the absorbance of the alpha-helical structures, and the increase in the absorbance of aggregated structures all have in common a multistep transition, the minor one centered at 45 degrees C and the major one around 59 degrees C. In contrast, a band that is tentatively assigned to loop structures displays these same minor and major transitions but at lower temperatures (39 and 52 degrees C, respectively). The transition, which occurs at 39-45 degrees C, is not associated with the appearance of aggregated structures. This transition may reflect a change in the tertiary structure of the protein. However, the final transition, which occurs at a higher temperature (52-59 degrees C), reflects unfolding and aggregation of the polypeptide chains. The Fourier transform infrared (FTIR) analysis suggests that PCS has a thermolabile region that unfolds first, some 7 degrees C below the main unfolding of the protein. We propose that this reflects the unfolding of the highly flexible loop segments, which in turn triggers the unfolding of the predominantly helical core structure of PCS.     

Mechanical unfolding of TNfn3: the unfolding pathway of a fnIII domain probed by protein engineering, AFM and MD simulation

Protein engineering Phi-value analysis combined with single molecule atomic force microscopy (AFM) was used to probe the molecular basis for the mechanical stability of TNfn3, the third fibronectin type III domain from human tenascin. This approach has been adopted previously to solve the forced unfolding pathway of a titin immunoglobulin domain, TI I27. TNfn3 and TI I27 are members of different protein superfamilies and have no sequence identity but they have the same beta-sandwich structure consisting of two antiparallel beta-sheets. TNfn3, however, unfolds at significantly lower forces than TI I27. We compare the response of these proteins to mechanical force. Mutational analysis shows that, as is the case with TI I27, TNfn3 unfolds via a force-stabilised intermediate. The key event in forced unfolding in TI I27 is largely the breaking of hydrogen bonds and hydrophobic interactions between the A' and G-strands. The mechanical Phi-value analysis and molecular dynamics simulations reported here reveal that significantly more of the TNfn3 molecule contributes to its resistance to force. Both AFM experimental data and molecular dynamics simulations suggest that the rate-limiting step of TNfn3 forced unfolding reflects a transition from the extended early intermediate to an aligned intermediate state. As well as losses of interactions of the A and G-strands and associated loops there are rearrangements throughout the core. As was the case for TI I27, the forced unfolding pathway of TNfn3 is different from that observed in denaturant studies in the absence of force.     

Searching for multiple folding pathways of a nearly symmetrical protein: temperature dependent phi-value analysis of the B domain of protein A

The B domain of protein A (BdpA) is a popular paradigm for simulating protein folding pathways. The discrepancies between so many simulations and subsequent experimental testing may be attributable to the protein being highly symmetrical: changing experimental conditions could perturb the subtle interplay between the effects of symmetry in the native structure and the effects of asymmetry from specific interactions in a given sequence. If the protein folds via multiple pathways, perturbations, such as temperature, denaturant concentration, and mutation, should change the flux of micro pathways, leading to changes in the bulk properties of the transition state. We tested this hypothesis by conducting a Phi-analysis of BdpA as a function of temperature from 25.0 degrees C to 60.0 degrees C. The Phi-values had no significant dependence on temperature and the values at 55.0 degrees C (denaturing conditions) are very similar to those at 25.0 degrees C (folding conditions), indicating the structure of the transition state does not significantly change although the experimental conditions are considerably altered. The results suggest that BdpA folds via a single dominant folding pathway.     

Temperature dependence of beta receptor, adenosine receptor, and sodium fluoride stimulated adenylate cyclase from turkey erythrocytes

The individual temperature dependencies of the process which control the activity of turkey erythrocyte adenylate cyclase have been determined. The temperature dependence of the fraction of activable cyclase units experiences a thermal transition at 24 degrees C for all three modes of enzyme activation: l-epinephrine, adenosine, and NaF. This thermal transition probably reflects the phase transition in the inner monolayer of the membrane which influences the behavior of the GTP regulatory unit which is involved in all three modes of enzyme activation. The "rate constant" of enzyme activation by adenosine reflects two thermal transitions, at 24 and at 35 degrees C; the apparent rate constant of cyclase activation by NaF activation experiences a transition only at 24 degrees C whereas the rate constant of the beta-receptor-bound agonist decreases monotonously with no "breaks" on the Arrhenium plot. Following the temperature dependence of the fluorescence intensity of dansylphosphatidylethanolamine embedded in both sides of the membrane and exclusively in the outer monolayer, one can assign the thermal transition of 24 degrees C to the inner monolayer and the other two transitions to the outer monolayer (10 and 35 degrees C). We interpret these results as follows. (a) The monomolecular rate constant characterizing the activation of cyclase by the precoupled adenosine receptor experiences both the transition at 24 and 35 degrees C, indicating that the latter may span the bilayer. (b) The bata receptor activates the cyclase units only in fluid areas since it can diffuse exclusively in the fluid areas of the membrane and is unable to interact with cyclase units in "frozen" areas. the linear dependence of the logarithm of the rate constant on 1/T for the bata receptor reflects the change of membrane fluidity as a function of temperature.     

Phase metastability and supercooled metastable state of diundecanoylphosphatidylethanolamine bilayers

Aqueous dispersons of L-alpha-phosphatidylethanolamine (PE) with identical saturated acyl chains are known to exhibit gel-state metastability. It is also known that the metastability in PE becomes more pronounced with decreasing acyl chain-length. In an attempt to study the metastable phase behavior of PE, we have synthesized diundecanoylphosphatidylethanolamine (diC11PE) and examined its polymorphic phase behavior. A single endothermic transition at 38 degrees C is detected between 10 and 55 degrees C by DSC for the nonheated sample of diC11PE in excess water. An immediate second heating scan done after cooling slowly of the same sample from the liquid-crystalline state shows a smaller endothermic transition at a lower temperature, 18 degrees C. However, the high-temperature transition at 38 degrees C can be detected, if the sample which has been heated above 38 degrees C is quench cooled from the liquid-crystalline to a temperature between 18 and 38 degrees C. Furthermore, two endothermic transitions at 18 and 38 degrees C and an exothermic transition at 19 degrees C are recorded for diC11PE after quench supercooling of the sample from the liquid-crystalline state to an appropriate temperature below 10 degrees C. The gel-state metastability of diC11PE can be most appropriately explained in terms of changes in interbilayer headgroup-headgroup interactions. It is suggested that the kinetically trapped supercooled metastable state may be a multilamellar structure with melted acyl chains but with strong interbilayer headgroup-headgroup interactions.     

Microaggregation of hormone-occupied epidermal growth factor receptors on plasma membrane preparations.

The rotational diffusion of the complexes of epidermal growth factor (EGF) with its specific receptor on plasma membrane vesicles prepared from human epidermoid carcinoma A431 cells was studied using the time-resolved polarization of phosphorescence of erythrosin-labeled hormone. The measured rotational correlation times of 16-20 microseconds at 4 degrees C are consistent with monomeric freely diffusing EGF receptor. Upon increasing the temperature to 37 degrees C, the rate of rotational diffusion slows down as evidenced by an increase in the correlation time to 75 microseconds. This finding suggests that small clusters of the occupied EGF receptor (microaggregation) form at the higher temperature, a property we have reported previously for occupied receptors on living A431 cells. Subsequent cooling of the membranes leads to a partial reversal of the microaggregation. We conclude that clustering of occupied EGF receptors can proceed at 37 degrees C in the absence of metabolic energy and external interactions, e.g. with components of the cytoskeleton, and thus reflects inherent properties of the receptor protein in its natural environment. A lag phase in the time course of microaggregation observed with the isolated membrane preparations may reflect cooperativity in the process of receptor association.     

Temperature-induced structural changes in putidaredoxin: a circular dichroism and UV-VIS absorption study

Putidaredoxin (Pdx) is an 11,400-Da iron-sulfur protein that sequentially transfers two electrons to the cytochrome P450cam during the enzymatic cycle of the stereospecific camphor hydroxylation. We report two transitions in the Pdx UV-VIS absorption and circular dichroism (CD) temperature dependencies, occurring at 16.3+/-0.5 degrees C and 28.4+/-0.5 degrees C. The 16.3 degrees C transition is attributed to the disruption of the hydrogen bonding of the active center bridging sulfur atom with cysteine 45 and alanine 46. The transition at 28.4 degrees C occurs exclusively in the Pdx(ox) at very nearly the same temperature as the earlier reported biphasicity in the redox potential. The formal potential temperature slope constancy reflects the relative stability of the concentration ratio of both oxidation states. The lower temperature transition affects both Pdx(red) and Pdx(ox) to a comparable extent, and their concentration ratio remains constant. In contrast, the 28.4 degrees C transition preferentially destabilizes Pdx(ox) thereby accelerating the formal potential negative shift and lower redox reaction entropy. There is evidence to suggest that disrupting hydrogen bonding of the iron ligating cysteines 45, 39 with residues threonine 47, serine 44, glycine 41, and serine 42 causes the 28.4 degrees C transition. The sensitivity of the UV-VIS absorption and CD spectroscopy to subtle structural protein backbone transitions is demonstrated.     

Mechanical unfolding of a titin Ig domain: structure of transition state revealed by combining atomic force microscopy,protein engineering and molecular dynamics simulations

Titin I27 shows a high resistance to unfolding when subject to external force. To investigate the molecular basis of this mechanical stability, protein engineering Phi-value analysis has been combined with atomic force microscopy to investigate the structure of the barrier to forced unfolding. The results indicate that the transition state for forced unfolding is significantly structured, since highly destabilising mutations in the core do not affect the force required to unfold the protein. As has been shown before, mechanical strength lies in the region of the A' and G-strands but, contrary to previous suggestions, the results indicate clearly that side-chain interactions play a significant role in maintaining mechanical stability. Since Phi-values calculated from molecular dynamics simulations are the same as those determined experimentally, we can, with confidence, use the molecular dynamics simulations to analyse the structure of the transition state in detail, and are able to show loss of interactions between the A' and G-strands with associated A-B and E-F loops in the transition state. The key event is not a simple case of loss of hydrogen bonding interactions between the A' and G-strands alone. Comparison with Phi-values from traditional folding studies shows differences between the force and "no-force" transition states but, nevertheless, the region important for kinetic stability is the same in both cases. This explains the correspondence between hierarchy of kinetic stability (measured in stopped-flow denaturant studies) and mechanical strength in these titin domains.     

Calorimetric investigations of the structural stability and interactions of colicin B domains in aqueous solution and in the presence of phospholipid bilayers

The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The calorimetric properties were compared with those of the isolated pore-forming fragment. The unfolding profile of the full-length toxin is consistent with two endothermic transitions. Whereas peak A (T(m) = 55 degrees C) most likely corresponds to the receptor/translocation domain, peak B (T(m) = 59 degrees C) is associated with the pore-forming domain. By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18 degrees C, respectively, due to proton exchange upon denaturation. The isolated pore-forming fragment unfolds at much higher temperatures (T(m) = 65 degrees C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation. In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B ( approximately 40%) or its pore-forming fragment ( approximately 80%). Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature. In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure. The heat of transition of membrane-associated colicin B (T(m) = 54 degrees C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions.     

Polytetrapeptide of elastin. Temperature-correlated elastomeric force and structure development

High molecular weight polytetrapeptide of elastin, (L.Val1-L.Pro2-Gly3-Gly4)n, was synthesized using activation of the (GGVP) permutation for polymerization. The temperature-dependence of aggregation was characterized as a function of concentration and the circular dichroism spectra were obtained in the 20 degrees to 70 degrees C temperature range. The latter showed an inverse temperature transition centered near 50 degrees C in which polypeptide order increased on raising the temperature. A concentration of 0.6 g of polytetrapeptide in 1 g of water was gamma irradiation cross-linked (20 Mrad) to form an elastomeric matrix. A study of the temperature-dependence of elastomeric force demonstrated a transition toward increased force on raising the temperature with a midpoint of the transition near 50 degrees C. Thus, there is a correlation between increase in intramolecular order and elastomeric force development. These results are compared to previous results on the polypentapeptide of elastin, (VPGVG)n and on an analog, (IPGVG)n, to demonstrate that the temperature of the transition is proportional to the hydrophobicity of the repeating unit. The point is noted that the elastomeric force development correlates better with intramolecular ordering than with intermolecular processes.     

Entropic elastic processes in protein mechanisms. I. Elastic structure due to an inverse temperature transition and elasticity due to internal chain dynamics

Numerous physical characterizations clearly demonstrate that the polypentapeptide of elastin (Val1-Pro2-Gly3-Val4-Gly5)n in water undergoes an inverse temperature transition. Increase in order occurs both intermolecularly and intramolecularly on raising the temperature from 20 to 40 degrees C. The physical characterizations used to demonstrate the inverse temperature transition include microscopy, light scattering, circular dichroism, the nuclear Overhauser effect, temperature dependence of composition, nuclear magnetic resonance (NMR) relaxation, dielectric relaxation, and temperature dependence of elastomer length. At fixed extension of the cross-linked polypentapeptide elastomer, the development of elastomeric force is seen to correlate with increase in intramolecular order, that is, with the inverse temperature transition. Reversible thermal denaturation of the ordered polypentapeptide is observed with composition and circular dichroism studies, and thermal denaturation of the crosslinked elastomer is also observed with loss of elastomeric force and elastic modulus. Thus, elastomeric force is lost when the polypeptide chains are randomized due to heating at high temperature. Clearly, elastomeric force is due to nonrandom polypeptide structure. In spite of this, elastomeric force is demonstrated to be dominantly entropic in origin. The source of the entropic elastomeric force is demonstrated to be the result of internal chain dynamics, and the mechanism is called the librational entropy mechanism of elasticity. There is significant application to the finding that elastomeric force develops due to an inverse temperature transition. By changing the hydrophobicity of the polypeptide, the temperature range for the inverse temperature transition can be changed in a predictable way, and the temperature range for the development of elastomeric force follows. Thus, elastomers have been prepared where the development of elastomeric force is shifted over a 40 degrees C temperature range from a midpoint temperature of 30 degrees C for the polypentapeptide to 10 degrees C by increasing hydrophobicity with addition of a single CH2 moiety per pentamer and to 50 degrees C by decreasing hydrophobicity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Parallel inhibition of active force and relaxed fiber stiffness by caldesmon fragments at physiological ionic strength and temperature conditions: additional evidence that weak cross-bridge binding to actin is an essential intermediate for force generation.

Previously we showed that stiffness of relaxed fibers and active force generated in single skinned fibers of rabbit psoas muscle are inhibited in parallel by actin-binding fragments of caldesmon, an actin-associated protein of smooth muscle, under conditions in which a large fraction of cross-bridges is weakly attached to actin (ionic strength of 50 mM and temperature of 5 degrees C). These results suggested that weak cross-bridge attachment to actin is essential for force generation. The present study provides evidence that this is also true for physiological ionic strength (170 mM) at temperatures up to 30 degrees C, suggesting that weak cross-bridge binding to actin is generally required for force generation. In addition, we show that the inhibition of active force is not a result of changes in cross-bridge cycling kinetics but apparently results from selective inhibition of weak cross-bridge binding to actin. Together with our previous biochemical, mechanical, and structural studies, these findings support the proposal that weak cross-bridge attachment to actin is an essential intermediate on the path to force generation and are consistent with the concept that isometric force mainly results from an increase in strain of the attached cross-bridge as a result of a structural change associated with the transition from a weakly bound to a strongly bound actomyosin complex. This mechanism is different from the processes responsible for quick tension recovery that were proposed by Huxley and Simmons (Proposed mechanism of force generation in striated muscle. Nature. 233:533-538.) to represent the elementary mechanism of force generation.     

Annular lipids determine the ATPase activity of a calcium transport protein complexed with dipalmitoyllecithin.

Pure complexes of dipalmitoyllecithin (DPL, 16:0) which Ca2+, Mg2+ dependent ATPase from sarcoplasmic reticulum are unusual in retaining significant ATPase activity down to about 30 degrees C, well below the transition temperature of the pure lipid at 41 degrees C. A minimum of about 35 lipid molecules per ATPase is required to maintain maximal ATPase activity, but the complexes are progressively and irreversibly inactivated at lower lipid to protein ratios. Complexes containing more than the minimum lipid requirement show very similar temperature profiles of activity about 30 degrees C over a wide range of lipid to protein ratios, up to 1500:1. Spin-label studies indicate that, at lipid to protein ratios of less than about 30 lipids per ATPase, no DPL phase transition can be detected, but at all higher ratios, a phase transition occurs at about 41 degrees C. In all of these complexes there are breaks in the Arrhenius plots of ATPase activity at 27--32 degrees C and at 37.5--38.5 degrees C. Experiments with perturbing agents, such as cholesterol and benzyl alcohol which have well-defined effects on the DPL phase transition, indicate that these breaks in the Arrhenius plots of ATPase activity cannot be attributed to a depressed and broadened phase transition in the lipids near the protein molecules. These results are interpreted as evidence for a phospholipid annulus of at least 30 lipid molecules with interact directly with the ATPase and cannot undergo a phase transition at 41 degrees C. This structural interaction of the ATPase with the annular DPL molecules has a predominant effect in determining the form of the temperature-activity profiles. However, the perturbation of the DPL phase transition does not extend significantly beyond the annulus since a phase transition which starts at 41 degrees C can be detected as soon as extraannular lipid is present in the complexes. We suggest that it may be a general feature of membrane structure that penetrant membrane proteins interact with their immediate lipid environment so as to cause only a minimal perturbation of the lipid bilayer.     

Differential scanning calorimetry of Cu,Zn-superoxide dismutase, the apoprotein, and its zinc-substituted derivatives

We have employed differential scanning calorimetry (DSC) to investigate the thermally induced unfolding of native Cu,Zn-superoxide dismutase (SOD), the apoprotein derived from native SOD, and the zinc-substituted derivatives of the apoprotein. We observe two overlapping melting transitions for native bovine SOD with heat capacity maxima at temperatures (Tm) of 89 and 96 degrees C when a scanning rate of 0.82 deg/min is employed. By contrast, the dithionite-reduced native SOD (which contains Cu+ rather than Cu2+) exhibits only a single transition at 96 degrees C. Significantly, we find that the concentration of O2 present in native SOD samples influences the relative magnitudes of the 89 and 96 degrees C peaks. Specifically, the lower temperature transition becomes less pronounced as the concentration of O2 in the sample decreases. On the basis of these observations, we propose that the lower temperature peak corresponds to the melting of the oxidized native protein, while the higher temperature peak reflects the melting of the reduced native protein, which forms spontaneously during the heating process. Our interpretation profoundly differs from that of Lepock et al. [Lepock, J.R., Arnold, L.D., Torrie, B.H., Andrews, B., & Kruuv, J. (1985) Arch. Biochem. Biophys. 241, 243-251], who have proposed that the low-temperature transition corresponds to the reduced form of the protein. We present evidence that suggests that their experiments were complicated by the presence of potassium ferrocyanide, which, in addition to reducing the cupric center, also perturbs the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Entropy and heat capacity of DNA melting from temperature dependence of single molecule stretching

When a single molecule of double-stranded DNA is stretched beyond its B-form contour length, the measured force shows a highly cooperative overstretching transition. We have measured the force at which this transition occurs as a function of temperature. To do this, single molecules of DNA were captured between two polystyrene beads in an optical tweezers apparatus. As the temperature of the solution surrounding a captured molecule was increased from 11 degrees C to 52 degrees C in 500 mM NaCl, the overstretching transition force decreased from 69 pN to 50 pN. This reduction is attributed to a decrease in the stability of the DNA double helix with increasing temperature. These results quantitatively agree with a model that asserts that DNA melting occurs during the overstretching transition. With this model, the data may be analyzed to obtain the change in the melting entropy DeltaS of DNA with temperature. The observed nonlinear temperature dependence of DeltaS is a result of the positive change in heat capacity of DNA upon melting, which we determine from our stretching measurements to be DeltaC(p) = 60 +/- 10 cal/mol K bp, in agreement with calorimetric measurements.     

Halothane, an inhalational anesthetic agent, increases folding stability of serum albumin

Inhalational anesthetic agents are known to alter protein function, but the nature of the interactions underlying these effects remains poorly understood. We have used differential scanning calorimetry to study the effects of the anesthetic agent halothane on the thermally induced unfolding transition of bovine serum albumin. We find that halothane (0.6-10 mM) stabilizes the folded state of this protein, increasing its transition midpoint temperature from 62 to 71 degrees C. Binding of halothane to the native state of serum albumin thus outweighs any non-specific interactions between the thermally unfolded state of serum albumin and halothane in this concentration range. Based on the average enthalpy change DeltaH for unfolding of 170 kcal/mol, the increase from 62 to 71 degrees C corresponds to an additional Gibbs energy of stabilization (DeltaDeltaG) due to halothane of more than 4 kcal/mol. Analysis of the dependence of DeltaDeltaG on halothane concentration shows that thermal unfolding of a bovine serum albumin molecule is linked to the dissociation of about one halothane molecule at lower halothane concentrations and about six at higher halothane concentrations. Serum albumin is the first protein that has been shown to be stabilized by an inhalational anesthetic.     

Effects of low temperature on contraction in papillary muscles from rabbit, rat, and hedgehog

During hibernation the body temperature may fall to only a few degrees above 0 degree C. The heart of the hedgehog continues to function whereas the hearts of nonhibernating mammals stop beating. The present study was performed to investigate and compare the mechanical responses to hypothermia in rabbits, rats, and hedgehogs. Isometric force was recorded from papillary muscles mounted in an organ bath and effects of hypothermia on the mechanical restitution curve were also compared. A reduction of bath temperature from 35 degrees C caused an increase in peak developed force. Maximum force was seen at 20 degrees C in the rabbit, 15 degrees C in the rat, and 10 degrees C in the hedgehog preparations. In all the species there was a similar prolongation of time to peak force and of time from peak to half-relaxation as temperature was lowered. An increase in resting force and after-contractions were recorded in the rabbit and rat muscles at temperatures below 15 and 10 degrees C, respectively. The rabbit and rat preparations became inexcitable at temperatures below 10 and 5 degrees C, respectively. The hedgehog papillary muscle, on the other hand, still contracted at 0 degree C and did not show increased resting force nor after-contractions. The results are consistent with the hypothesis that there is a calcium overload in cardiac cells from rabbit and rat at low temperatures but there is no calcium overload in the hedgehog muscle during hypothermia.     

Thermal response of Langmuir-Blodgett films of dipalmitoylphosphatidylcholine studied by atomic force microscopy and force spectroscopy

The topographic evolution of supported dipalmitoylphosphatidylcholine (DPPC) monolayers with temperature has been followed by atomic force microscopy in liquid environment, revealing the presence of only one phase transition event at approximately 46 degrees C. This finding is a direct experimental proof that the two phase transitions observed in the corresponding bilayers correspond to the individual phase transition of the two leaflets composing the bilayer. The transition temperature and its dependency on the measuring medium (liquid saline solution or air) is discussed in terms of changes in van der Waals, hydration, and hydrophobic/hydrophilic interactions, and it is directly compared with the transition temperatures observed in the related bilayers under the same experimental conditions. Force spectroscopy allows us to probe the nanomechanical properties of such monolayers as a function of temperature. These measurements show that the force needed to puncture the monolayers is highly dependent on the temperature and on the phospholipid phase, ranging from 120+/-4 pN at room temperature (liquid condensed phase) to 49+/-2 pN at 65 degrees C (liquid expanded phase), which represents a two orders-of-magnitude decrease respective to the forces needed to puncture DPPC bilayers. The topographic study of the monolayers in air around the transition temperature revealed the presence of boundary domains in the monolayer surface forming 120 degrees angles between them, thus suggesting that the cooling process from the liquid-expanded to the liquid-condensed phase follows a nucleation and growth mechanism.     

Comparative study of an adenosine triphosphatase trigger-fused lipid vesicle and other vesicle forms of dimyristoylphosphatidylcholine

Several known forms of bilayer vesicles of dimyristoylphosphatidylcholine exhibit the gel to liquid-crystalline phase transition in the temperature range convenient for membrane enzyme reconstitution studies. This warrants a systematic investigation of their physical characteristics and their phase transition behaviors. We have employed electron microscopy, gel chromatography, 31P nuclear magnetic resonance, differential scanning microcalorimetry, and fluorescence spectroscopy to determine several physical parameters of the limiting size microvesicle (260 +/- 40 A), the larger vesicle form (900 +/- 100A) of Enoch and Strittmatter [Enoch, H. G., & Strittmatter, P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 145], the multilamellar vesicle, and, in particular, an ATPase-trigger-fused macrovesicle (950 +/- 200 A). This latter vesicle form was produced by a spontaneous fusion of the complex of the plasma membrane ATPase of Schizosaccharomyces pombe and the lipid microvesicles at a low ratio of enzyme to vesicle concentrations, and at a low temperature (around 10 degrees C). The ATPase-trigger-fused vesicles are unilamellar and have an intact ionic permeation barrier at 30 degrees C and a gel to liquid-crystalline transition temperature at 24.4 degrees C with a transition heat of 5.64 kcal/mol. Thus, this vesicle form should be a valuable tool for studying possible proton-pumping activity of this ATPase. In contrast to data found in the literature, which show lack of the pretransition for unilamellar microvesicles, we have observed the pretransition around 15 degrees C for all the vesicle forms examined. Moreover, the transition widths of unilamellar vesicles are much broader than those of the multilamellar vesicles, suggesting that in the latter system interlayer interactions may contribute to the cooperativity of the transition.     

Phase behavior of membranes reconstituted from dipentadecanoylphosphatidylcholine and the Mg2+-dependent, Ca2+-stimulated adenosinetriphosphatase of sarcoplasmic reticulum: evidence for a disrupted lipid domain surrounding protein

A new method was used for reconstituting active sodium deoxycholate solubilized Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum. Removal of the detergent by dialysis at the pretransition temperature of the pure lipid (22 degrees C) favored the formation of sheet-like structures with a lipid and protein content close to that of the detergent-solubilized sample. Freeze-fracture electron micrographs revealed the Ca2+-ATPase to be organized in rows corresponding to the typical banded pattern seen in low-temperature freeze-fracture micrographs of pure lipid bilayers. Incubation of the sheetlike structures at a temperature (38 degrees C) above the pure lipid main phase transition (33.5 degrees C) caused closure of the sheets into vesicles displaying homogeneous intramembranous particle distributions, at least for membranes containing less than 150 lipids per Ca2+-ATPase. However, in membranes of higher lipid content, free lipid patches were seen both above and below the lipid phase transition. By use of high-sensitivity differential scanning calorimetry, three classes of excess heat capacity peaks were observed in the vesiculated samples. A broadened "free lipid" peak occurred for samples containing between 550 and 200 lipids per protein (Tm = 33.5 degrees C, as for the order-disorder transition in pure lipid vesicles). Between 200 and 150 lipids per Ca2+-ATPase, a broad shoulder became apparent in the range of 29-32 degrees C. Below 150 lipids per Ca2+-ATPase, a peak at 26-28 degrees C became increasingly prominent with lower lipid content. At a lipid to protein ratio of about 30, no peaks in heat capacity were observed. The temperature dependence of diphenylhexatriene fluorescence anisotropy revealed a similar pattern of membrane phase behavior, except that a phase transition was detected at 33.5 degrees C in all membranes studied. On the basis of these observations, we propose that the Ca2+-ATPase is surrounded by a "lipid annulus" of motionally inhibited lipid molecules that do not contribute to a calorimetrically detectable phase transition. Beyond the annulus, "secondary domains" of disrupted lipid packing account for the peak at 26-28 degrees C and the 29-32 degrees C shoulders. At high lipid to protein ratios, the secondary domains coexist with protein-free, lipid-bilayer patches, which account for the peak at 33.5 degrees C.