Julian M. Sturtevant

Incorporation of saturated fatty acids into phosphatidylcholine bilayers

The transition temperature of dipalmitoylglycerophosphocholine in multi-lamellar aqueous suspensions, as observed by high-sensitivity differential scanning calorimetry, is raised from 41.4 to 61.5 degrees C by addition of palmitic acid at a mole fraction of 0.67. It appears that the fatty acid chains pack in the hexagonal lattice with the lipid chains in a one-to one ratio, thereby eliminating the destabilizing crowding of the phosphatidylcholine head groups. A similar effect on dilauroylglycerophosphocholine is produced by lauric acid. The stabilizing effect is not produced in full measure by acids of different chain lengths, nor by alcohols or saturated hydrocarbons of the same chain length.

Characterization of the plasma membrane of Mycoplasma laidlawii. VII. Phase transitions of membrane lipids

Abstract Use of a very sensitive differential calorimeter has revealed two major regions of heat absorption in purified plasma membranes of Mycoplasma laidlawii. These consist of a rapidly increasing rate of heat absorption showing an inflection point at approx. 40°; this is followed at higher temperatures by a slightly decreased rate of heat uptake. Within this latter region it is barely possible to distinguish a second inflection point. These transitions were found also to be properties of whole cells. Experimental results presented in this paper support the conclusion that the first region of heat absorption is due to a reversible transition in the lipid component of the membrane, and that the second region is due to an irreversible denaturation of the protein component. These results also provide evidence that the lipid transition is essentially unaffected by the protein component of the membrane. The calorimetric transition curves indicate that the apparent heat capacity of the membrane lipids increases during the transition. This most likely arises from new modes of freedom available to the fatty acid hydrocarbon chains in the melted state. By means of a viable cell titer taken before and after cells were submitted to a calorimetric run extending to 40°, the lipid transition was demonstrated unambiguously to be a property of living cells. The Tm of the lipid transition was found to be a function of the conditions of growth of the cells. In all cases studied the temperature at which the cells grew lies within the range of the lipid transition. Results suggest that the cells attempt to regulate their position in the lipid transition.

High-Sensitivity Differential Scanning Calorimetry in the Study of Biomembranes and Related Model Systems

Differential scanning calorimetry (DSC) currently finds wide application in studies of the thermotropic behavior of lipids in biological membranes and related systems (Chapman, 1975a,b; Scheidler and Steim, 1975). Almost without exception these studies have employed calorimetric equipment which is limited to very small sample sizes and relatively high scan rates, so that only low sensitivity is realized with the aqueous solutions and suspensions which are of interest in biology. The recent development of scanning calorimeters especially adapted for applications involving relatively dilute solutions and suspensions (Gill and Beck, 1965; Tsong et al., 1970; Jackson and Brandts, 1970; Ross and Goldberg, 1974; Grubert and Ackermann, 1974; Privalov et al., 1975; and Suurkuusk et al., 1976) opens up the possibility of more searching and quantitative studies of these systems. The present chapter outlines the fundamental principles of DSC, and discusses several recent applications of high-sensitivity DSC to membranes and related systems, with particular reference to the most highly developed scanning calorimeter currently available, the one designed by Privalov (Privalov et al., 1975).

Thermotropic behavior of binary mixtures of diplamitoylphosphatidylcholine and glycosphingolipids in aqueous dispersions

The thermotropic behavior of mixtures of dipalmitoylphosphatidylcholine (DPPC) with natural glycosphingolipids (galactosylceramide, phrenosine, kerasine, glucosylceramide, lactosylceramide, asialo-GM1, sulfatide, GM3, GM1, GD1a, GT1b) in dilute aqueous dispersions were studied by high sensitivity differential scanning calorimetry over the entire composition range. The pretransition of DPPC is abolished and the cooperativity of the main transition decreases sharply at mole fractions of glycosphingolipids below 0.2. All systems exhibit non-ideal temperature-composition phase diagrams. The mono- and di-hexosylceramides are easily miscible with DPPC when the proportion of glycosphingolipids in the system is high. A limited quantity (1-6 molecules of DPPC per molecule of glycosphingolipid (GSL) can be incorporated into a homogeneously mixed lipid phase. Domains of DPPC, immiscible with the rest of a mixed GSL-DPPC phase that shows no cooperative phase transition, are established as DPPC exceeds a certain proportion in the system. One negative charge (sulfatide) or four neutral carbohydrate residues (asialo-GM1) in the oligosaccharide chain of the glycosphingolipids results in phase diagrams exhibiting coexistence of gel and liquid phases over a broad temperature-composition range. Systems containing gangliosides show complex phase diagrams, with more than one phase transition. However, no evidence for phase-separated domains of pure ganglioside species is found. The thermotropic behavior of systems containing DPPC and glycosphingolipids correlates well with their interactions in mixed monolayers at the air/water interface.

Thermotropic behavior of retinal rod membranes and dispersions of extracted phospholipids

SummaryHigh sensitivity, differential scanning calorimetry studies of vovine retinal rod outer segment (ROS) disk membranes and aqueous dispersions of the extracted ROS phospholipids have been performed. ROS disk membranes were found to exhibit a broad peak of excess heat capacity with a maximum at less than about 3°C, ascribable to a gel-to-liquid crystalline phase transition of traction of the phospholipids. A similar thermotropic transition was observed for aqueous dispersions of the total extracted and purified ROS phospholipids. Comparison of the results obtained for the dispersion of total ROS phospholipids to those of the purified head group fractions. suggests that the thermotropic behavior reffects a gel-to-liquid crystalline transition, leading to lateral phase separation, involving those phosphatidylcholine (PC) molecules containing saturated fatty acylchains, possibley together with the highest melting ROS phosphatidylethanolamine (PE) and phosphatidylserine (PS) components. The interpretation of the thermal behavior of the ROS disk membranes depends on whether the transition is assumed to derive from the ROS PC and/or PE/PS fractions, and whether the transbilayer arrangement of the ROS phospholipids is assumed to be symmetric or asymmetric. The calorimetric data can be simply explained in terms of an asymmetric distribution of the major ROS disk membrane phospholipids (G.P. Miljanich et al.,J. Membrane Biol.60:249–255, 1981). In this case, the transition would arise from the PE/PS fractions in the outer ROS disk membrane monolyer, and the anticipated transition from the PC in the inner monolayer would be broadened due to interaction with cholesterol. For the ROS membranes at higher temperatures, two additional, irreversible transitions are observed at 57 and 72°C, corresponding to the thermal denauturation of opsin and rhodopsin, respectively.

Sidechain interactions in parallel β sheets: the energetics of cross-strand pairings

BACKGROUND Both backbone hydrogen bonding and interactions between sidechains stabilize beta sheets. Cross-strand interactions are the closest contacts between the sidechains of a beta sheet. Here we investigate the energetics of cross-strand interactions using a variant of the B1 domain of immunoglobulin G (IgG) binding protein G (beta1) as our model system. RESULTS Pairwise mutations of polar and nonpolar residues were made at a solvent-exposed site between the two central parallel beta strands of beta1. Both stabilizing and destabilizing interactions were measured. The greatest stabilizations were observed for charge-charge interactions. Our experimental study of sidechain interactions correlates with statistical preferences: residue pairs for which we measure stabilizing interaction energies occur together frequently, whereas destabilizing pairs are rarely observed together. CONCLUSIONS Sidechain interactions modulate the stability of beta sheets. We propose that cross-strand sidechain interactions specify correct strand register and ordering through the energetic benefit of optimally arranged pairings.

The thermodynamic effects of protein mutations

Abstract The development of cloning procedures has made possible a rapid increase in the appearance of reports on the thermodynamic effects of mutations in proteins. It will be helpful if the availability of excellent calorimetric instrumentation leads to an increase in the fraction of these studies directed toward the determination of enthalpies and entropies as well as free energies.

Significant discrepancies between van't Hoff and calorimetric enthalpies. III.

The study of a wide variety of reversible reactions in solution indicates that the enthalpy, DeltaH(vH), which controls the temperature variation of the equilibrium constant for a reaction, can seldom, if ever, be taken to be independent of the temperature. It is also found in most cases that the values for DeltaH(vH), properly evaluated as varying with the temperature, differ significantly from the values for the enthalpy, DeltaH(cal), determined by direct calorimetry under the same experimental conditions. In a continuing search for reactions which show agreement between DeltaH(vH) and DeltaH(cal), we have studied by isothermal titration calorimetry the reactions of heptylamine with heptanoic acid in dodecane solution and of alpha-cyclodextrin with sodium heptanoate in aqueous solution.

A calorimetric examinations of stable and fusing lipid bilayer vesicles

Mixed lipid samples containing dimyristoylglycerophosphocholine and small amounts of myristic acid were examined calorimetrically. Examination of multilamellar and small vesicle samples indicated that upon heating small vesicles combine to form more extended structures. An exothermic peak (at 19 . 5 degrees C) can be associated with the structural transformation. The enthalpy for this process, which may be interpreted as vesicle-vesicle fusion, is found to be approx.--2 kcal/mol.

Application of flow calorimetry to the determination of the enthalpies of mixing of organic liquids

Abstract A flow modification of the Beckman Model 190 Microcalorimeter is described with which the enthalpies of mixing of pairs of liquids can be very conveniently and accurately measured. The enthalpies of mixing of ten pairs of liquids at 25 °C are reported, composition ranges at least as wide as volume fraction 0.05 to 0.95 of one component being covered. Of the systems studied, only cyclohexane + carbon tetrachloride and n-octane + iso-octane were found to conform within experimental error to the strict requirements of Scatchards description of regular solutions. The systems cyclohexane + cis-decalin and cyclohexane + trans-decalin are endothermic over only part of the composition range, changing to exothermic in decalin-rich mixtures; the two decalins mix exothermically throughout the composition range. Cyclohexane + n-hexane and cyclohexane + n-heptane show rather marked deviations from symmetric behavior. The remaining systems, benzene + toluene, toluene + o-xylene and o-xylene + p-xylene, though differing widely in enthalpies of mixing, exhibit only slight deviations from regular behavior. The results confirm the Scatchard-Hildebrand equation in suggesting that enthalpies of mixing in cases such as those studied here are best presented in the form of values of ΔQ >φ 1 >φ 2 given as a function of >φ1. In this expression, ΔQ is the enthalpy of mixing per unit volume and >φ1 and >φ2 are the volume fractions of the components.