Rodney L. Biltonen

The use of differential scanning calorimetry as a tool to characterize liposome preparations

Abstract Differential scanning calorimetry (DSC) measures the temperature-dependence of the excess heat capacity of a system due to thermal phase transitions. Heat capacity curves of liposomes that undergo such transitions contain information on the enthalpy and entropy of these transitions. Being sensitive to both the chemical composition of these liposomes and their physical state (especially size), DSC can serve as a powerful tool for quality control of liposomes. If calorimetric criteria are appropriately established using a proper reference lipid sample, the quality and stability of a liposomal preparation which has a distinct thermogram can be assessed by DSC.

Lipid bilayer heterogeneities and modulation of phospholipase A2 activity.

The regulation of phospholipase A2 (PLA2) activity toward synthetic vesicular substrates is a model for the modulation c enzyme function by biological membranes. PLA2s catalytic rate toward membrane phospholipids can be modified by several order of magnitude by altering the membranes composition and structure. The physical basis of this sensitivity is the subject of thi report. The results described here imply that the salient features of membrane-structure which modulate PLA2 activity include compositional phase separation; membrane curvature and, possibly, curvature-associated defects; and dynamic product inhibition due to limitations imposed by the rate of lateral diffusion of lipid in the membrane. Furthermore, it is shown that the effects of membrane structure on the catalytic rate are not exerted merely by enhancing association of PLA2 with the membrane surface: a membrane-bound inactive state is spectroscopically identified. Finally, these results are discussed in the context of some published models for the role of membrane structure in the regulation of membrane-bound enzymes.

A statistical-thermodynamic view of cooperative structural changes in phospholipid bilayer membranes: their potential role in biological function

It is a great honor to receive the Huffman Memorial Award from the Calorimetry Conference. Such an honor was certainly not on my list of expectations when I first became interested in calorimetric experimentation over 20 years ago. I thank the conference for this honor and the many of you who have aided and encouraged the work which this award recognizes. In 1969, I had the opportunity to spend three months in the laboratories of the late Stig Sunner and Ingemar Wadsii at the University of Lund, Sweden. It was during that visit that I first became acquainted with state-of-the-art calorimetric equipment. That visit provided me the opportunity to explore from a practical perspective the many possibilities of applying calorimetric techniques to probe the thermodynamic details of questions of biological interest. My interest in, and motivation for, using calorimetric techniques has always been driven by specific thermodynamic questions related to biological systems. The original questions were related to understanding the thermodynamic mechanism of unfolding of globular proteins in aqueous solution. In the early sixties these thermally induced unfolding transitions were assessed by spectroscopic changes primarily and calculation of thermodynamic quantities associated with the process required that the two-state approximation (i.e. the protein existed in one of only two thermodynamic states) be valid, an assumption which had no previous means of being proven. Additionally, in 1964 Brandts(‘**) put forth a phenomenological

Estimation of molecular averages and equilibrium fluctuations in lipid bilayer systems from the excess heat capacity function

It is demonstrated that the bilayer partition function can be numerically obtained from scanning calorimetric data without assuming a particular model for the gel-liquid crystalline transition. From this partition function, the enthalpy, entropy and volume changes accompanying the transition can be calculated. In the limit of very large systems, the method of the grand partition function allows calculation of cluster model distribution functions from which average sizes of gel and liquid-crystal clusters, cluster densities and equilibrium fluctuations are obtained. These results indicate that the main transition in phospholipid bilayers proceeds through the formation of clusters and that these clusters are not static domains but highly fluctuating entities. These fluctuations in cluster size are approximately equal to the average cluster size and give rise to localized density and volume fluctuations. The magnitude of these fluctuations is affected by the radius of curvature of the bilayer and by the addition of small molecular weight compounds to the system.

Monte Carlo Simulation of Two-Component Bilayers: DMPC/DSPC Mixtures*

In this paper, we describe a relatively simple lattice model of a two-component, two-state phospholipid bilayer. Application of Monte Carlo methods to this model permits simulation of the observed excess heat capacity versus temperature curves of dimyristoylphosphatidylcholine (DMPC)/distearoylphosphatidylcholine (DSPC) mixtures as well as the lateral distributions of the components and properties related to these distributions. The analysis of the bilayer energy distribution functions reveals that the gel-fluid transition is a continuous transition for DMPC, DSPC, and all DMPC/DSPC mixtures. A comparison of the thermodynamic properties of DMPC/DSPC mixtures with the configurational properties shows that the temperatures characteristics of the configurational properties correlate well with the maxima in the excess heat capacity curves rather than with the onset and completion temperatures of the gel-fluid transition. In the gel-fluid coexistence region, we also found excellent agreement between the threshold temperatures at different system compositions detected in fluorescence recovery after photobleaching experiments and the temperatures at which the percolation probability of the gel clusters is 0.36. At every composition, the calculated mole fraction of gel state molecules at the fluorescence recovery after photobleaching threshold is 0.34 and, at the percolation threshold of gel clusters, it is 0.24. The percolation threshold mole fraction of gel or fluid lipid depends on the packing geometry of the molecules and the interchain interactions. However, it is independent of temperature, system composition, and state of the percolating cluster.

Lipid lateral heterogeneity in phosphatidylcholine/phosphatidylserine/diacylglycerol vesicles and its influence on protein kinase C activation

To test the hypothesis that the activation of protein kinase C (PKC) is influenced by lateral heterogeneities of the components of the lipid bilayer, the thermotropic phase behavior of dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO) vesicles was compared with the activation of PKC by this system. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to monitor the main transition (i.e., the gel-to-fluid phase transition) as a function of mole fraction DO (chi(DO)) in DMPC/DO, DMPS/DO, and [DMPC/DMPS (1:1, mol/mol)]/DO multilamellar vesicles (MLVs). In each case, when chi(DO) < or approximately 0.3, DO significantly broadened the main transition and shifted it to lower temperatures; but when chi(DO) > approximately 0.3, the main transition became highly cooperative, i.e., narrow, again. The coexistence of overlapping narrow and broad transitions was clearly evident in DSC thermograms from chi(DO) approximately 0.1 to chi(DO) approximately 0.3, with the more cooperative transition growing at the expense of the broader one as chi(DO) increased. FTIR spectroscopy, using analogs of DMPC and DMPS with perdeuterated acyl chains, showed that the melting profiles of all three lipid components in [DMPC/DMPS (1:1, mol/mol)]/DO MLVs virtually overlay when chi(DO) = 0.33, suggesting that a new type of phase, with a phospholipid/DO mole ratio near 2:1, is formed in this system. Collectively, the results are consistent with the coexistence of DO-poor and DO-rich domains throughout the compositions chi(DO) approximately 0.1 to chi(DO) approximately 0.3, even at temperatures above the main transition. Comparison of the phase behavior of the binary mixtures with that of the ternary mixtures suggests that DMPS/DO interactions may be more favorable than DMPC/DO interactions in the ternary system, especially in the gel state. PKC activity was measured using [DMPC/DMPS (1:1, mol/mol)]/DO MLVs as the lipid activator. At 35 degrees C (a temperature above the main transition of the lipids), PKC activity increased gradually with increasing chi(DO) from chi(DO) approximately 0.1 to chi(DO) approximately 0.4, and activity remained high at higher DO contents. In contrast, at 2 degrees C (a temperature below the main transition), PKC activity exhibited a maximum between chi(DO) approximately 0.1 and chi(DO) approximately 0.3, and at higher DO contents activity was essentially constant at 20-25% of the activity at the maximum. We infer from these results that the formation of DO-rich domains is related to PKC activation, and when the lipid is in the gel state, the coexistence of DO-poor and DO-rich phases also contributes to PKC activation.

Phospholipase A2 activity and physical properties of lipid-bilayer substrates.

Publisher Summary This chapter proposes a rational strategy for a methodological study of phospholipase (PLA) activity by combining insight from a parallel set of theoretical calculations and experimental measurements, with the aim of unraveling details of the relationship between the physical properties on different scales of the substrate, on the one hand, and the activity of particularly PLA 2 , on the other. The chapter reviews the relevant physical properties of lipid bilayers because these are controlled by the underlying thermodynamics and phase equilibria. Particular attention is paid to in-plane phase transitions and dynamic lateral bilayer organization in terms of nanoscale lipid domains and microheterogeneity. The effects of non-bilayer-forming lipids are described in the chapter with reference to the possible influence on the substrate of the products of the hydrolysis. It provides the necessary background information on the hydrolysis of lipids by PLA 2 and how it can be monitored. The chapter describes the data for the dependence of the PLA 2 activity on the composition of the substrate and presents a comparison between experimental and theoretical results that provides strong evidence for a correlation between lipid-bilayer dynamic microheterogeneity and enzyme activity.

Altered affinity of CBFβ-SMMHC for Runx1 explains its role in leukemogenesis

Chromosomal translocations involving the human CBFB gene, which codes for the non-DNA binding subunit of CBF (CBFβ), are associated with a large percentage of human leukemias. The translocation inv(16) that disrupts the CBFB gene produces a chimeric protein composed of the heterodimerization domain of CBFβ fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (CBFβ-SMMHC). Isothermal titration calorimetry results show that this fusion protein binds the Runt domain from Runx1 (CBFα) with higher affinity than the native CBFβ protein. NMR studies identify interactions in the CBFβ portion of the molecule, as well as the SMMHC coiled-coil domain. This higher affinity provides an explanation for the dominant negative phenotype associated with a knock-in of the CBFB-MYH11 gene and also helps to provide a rationale for the leukemia-associated dysregulation of hematopoietic development that this protein causes.

[14] Microcalorimetry for biological chemistry: Experimental design, data analysis, and interpretation☆

Publisher Summary This chapter discusses the design of specific types of calorimetric experiments. The basic calorimetric experiment consists of mixing two solutions that contain reactive components. The success of a calorimetric experiment depends upon a number of factors, but perhaps the most commonly ignored is the achievement of thermal equilibrium of the solutions prior to mixing. It is important to realize that during the calorimetric experiment temperature changes on the order of l0 –4 degree or less are being measured. In the batch-type calorimeter, the loading of the solutions into the reaction vessels requires that the calorimetric heat sink and air bath be exposed to the environment, thus disturbing the existing equilibrium among the cells, heat sink, and bath. The time required to achieve satisfactory thermal equilibrium after the calorimeter is loaded depends upon the magnitude of the heat effect being measured. In the flow calorimeter, thermal equilibrium between the solutions and the calorimeter heat sink is achieved by flowing the solution through one or more heat exchangers that are in equilibrium with the heat sink. In the isoperibol and isothermal calorimeters, the solutions are equilibrated within the calorimeter prior to mixing.

Monte Carlo simulations of membranes: phase transition of small unilamellar dipalmitoylphosphatidylcholine vesicles.

Publisher Summary Monte Carlo (MC) methods have proved to be useful with model membrane systems, making it possible to simulate the fluctuating membrane conformations and calculate membrane properties and relate them to experimental observables. Correlations between particular conformational properties and complex membrane functions would aid in the interpretation of the functions and provide the basis for further experimental work. In any case, quantitative agreement between calculation and experiment is essential. This chapter describes a simple model for the gel-to-liquid crystalline transition of lipid bilayers. Bilayer membranes composed of a single lipid undergo a gel-to-liquid crystalline transition at a temperature defined by the chemical nature of the lipid, the degree of hydration, and basic structure of the membrane. This transition has been described extensively for bilayer membranes made of dipalmitoylphosphatidylcholine (DPPC). The approach is to introduce as much experimental information as possible into the calculation, assume values for a minimal number of additional parameters, and then test the results quantitatively with experimental data. The chapter reviews other MC simulations of one- and two-component membrane systems and discusses the computational methods applied.