F. Ross Hallett

Osmotically Induced Shape Changes of Large Unilamellar Vesicles Measured by Dynamic Light Scattering

Static and dynamic light scattering measurements have been used to characterize the size, size distribution, and shape of extruded vesicles under isotonic conditions. Dynamic light scattering was then used to characterize osmotically induced shape changes by monitoring changes in the hydrodynamic radius (R(h)) of large unilamellar vesicles (LUVs). These changes are compared to those predicted for several shapes that appear in trajectories through the phase diagram of the area difference elasticity (ADE) model (. Phys. Rev. E. 52:6623-6634). Measurements were performed on dioleoylphosphatidylcholine (DOPC) vesicles using two membrane-impermeant osmolytes (NaCl and sucrose) and a membrane-permeant osmolyte (urea). For all conditions, we were able to produce low-polydispersity, nearly spherical vesicles, which are essential for resolving well-defined volume changes and consequent shape changes. Hyper-osmotic dilutions of DOPC vesicles in urea produced no change in R(h), whereas similar dilutions in NaCl or sucrose caused reductions in vesicle volume resulting in observable changes to R(h). Under conditions similar to those of this study, the ADE model predicts an evolution from spherical to prolate then oblate shapes on increasing volume reduction of LUVs. However, we found that DOPC vesicles became oblate at all applied volume reductions.

Interaction of the 18.5-kD isoform of myelin basic protein with Ca2+-calmodulin: Effects of deimination assessed by intrinsic Trp fluorescence spectroscopy, dynamic light scattering, and circular dichroism

The effects of deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) on its binding to calmodulin (CaM) have been examined. Four species of MBP were investigated: unmodified recombinant murine MBP (rmMBP‐Cit0), an engineered protein with six quasi‐citrullinyl (i.e., glutaminyl) residues per molecule (rmMBP‐qCit6), human component C1 (hMBP‐Cit0), and human component C8 (hMBP‐Cit6), both obtained from a patient with multiple sclerosis (MS). Both rmMBP‐Cit0 and hMBP‐Cit0 bound CaM in a Ca2+‐dependent manner and primarily in a 1:1 stoichiometry, which was verified by dynamic light scattering. Circular dichroic spectroscopy was unable to detect any changes in secondary structure in MBP upon CaM‐binding. Inherent Trp fluorescence spectroscopy and a single‐site binding model were used to determine the dissociation constants: Kd = 144 ± 76 nM for rmMBP‐Cit0, and Kd = 42 ± 15 nM for hMBP‐Cit0. For rmMBP‐qCit6 and hMBP‐Cit6, the changes in fluorescence were suggestive of a two‐site interaction, although the dissociation constants could not be accurately determined. These results can be explained by a local conformational change induced in MBP by deimination, exposing a second binding site with a weaker association with CaM, or by the existence of several conformers of deiminated MBP. Titration with the collisional quencher acrylamide, and steady‐state and lifetime measurements of the fluorescence at 340 nm, showed both dynamic and static components to the quenching, and differences between the unmodified and deiminated proteins that were also consistent with a local conformational change due to deimination.

Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids

Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes. Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins. Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution. The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E. coli polar lipid by extrusion. Liposomes prepared from E. coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Youngs elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy. At high concentrations, NaCl caused aggregation of E. coli lipid liposomes that precluded detailed interpretation of light scattering data. Proteoliposomes and liposomes prepared from E. coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte. These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E. coli lipid vesicles.

Aging dynamics in gelatin gel microstructure

Gelatin is a biological polymer that forms thermoreversible gels. Even after formation, the structure of the gel is not static and continues to evolve and change because of the instability of the low energy interactions that connect the gel network. Rheological and dynamic light scattering (DLS) measurements were used to observe the development and evolution of the gel network structure during gelation. Rheological measurements were used to monitor the rate of increase in the number and strength of elastically effective chains as the network forms. Two power law scaling regions were observed; one where the aggregation process dominates and a second, where the rearrangement process dominates. DLS suggested that there were three evolving levels of structure in the gel network: small clusters, large groups of clusters and very large assemblies. For small clusters, the correlation length decreased as the concentration increased. For the large groups, increasing the setting temperature increased the maximum correlation length. Scattered intensity data suggested that large groups lose their integrity as the gel network rearranges.

The characterization of small emulsion droplets made from milk proteins and triglyceride oil

Measurements have been made of the light scattering properties of emulsion droplets produced using a Microfluidizer. The droplet size distributions were measured by both Integrated Light Scattering (ILS) over a wide range of scattering angles, and by Dynamic Light Scattering (DLS) at a fixed angle of 90°. From the former, it was possible to derive number distributions, whereas the latter was used to give intensity distributions of the droplets. The calculated distributions from light scattering were also compared with samples studied by transmission electron microscopy. A range of samples was studied, including whole milks, and emulsions prepared from soya oil and stabilized by sodium caseinate; fractions containing the smaller droplets from these emulsions were also collected and studied. The results confirmed that it was possible to isolate a population of small ( < 100 nm) particles from microfluidized milk or from the emulsions; larger particles were also present. Both ILS and electron microscopy confirmed these results. DLS also confirmed the presence of the small particles in the emulsions, but principally was useful in the analysis of the separated fractions. To improve the sensitivity of the method, it is necessary to use non-linear channel times in DLS experiments.

Modulation of myelin basic protein-induced aggregation and fusion of liposomes by cholesterol, aliphatic aldehydes and alkanes

The effect of cholesterol on myelin basic protein-induced aggregation of zwitterionic phospholipid vesicles was studied by turbidimetry, quasi-elastic light scattering and centrifugation techniques. Without cholesterol, the degree of vesicle aggregation caused by myelin basic protein is relatively low and is only slightly increased using cholesterol concentrations up to approx. 25-30 mol%. When the cholesterol content in the bilayer exceeds approx. 30 mol%, there is a dramatic increase in the susceptibility of the vesicles to aggregation in the presence of myelin basic protein. Palmitoyl aldehyde and eicosane, substances resembling products of lipid degradation, increase myelin basic protein promoted fusion of vesicles. The fusion is accompanied by increased leakage of entrapped carboxyfluorescein. In the presence of cholesterol, myelin basic protein-induced fusion of the liposomes becomes much more sensitive to the presence of aliphatic aldehydes or alkanes. The results suggest that cholesterol has an important role in promoting membrane adhesion in biological systems but these structures become unstable in the presence of small amounts of products of lipid degradation. The findings have important implications to the understanding of the stability of the myelin membrane.

Particle size analysis by dynamic light scattering

Abstract Dynamic light scattering is an established technique for measuring the average size and size distribution of particles in a suspension. The technique has the advantage of being fast and non-invasive, but it does require low particle concentrations. As well, dynamic light scattering results are often open to misinterpretation if one is unaware of the state of the sample and the method of data analysis. The following discussion reviews some of the basic concepts of dynamic light scattering and outlines some of the pitfalls that are often encountered in data interpretation. A modification of dynamic light scattering, diffusing wave spectroscopy, can be used to obtain approximate size information at higher particle concentrations. The fundamentals of this new technique are summarized.

Quasi-elastic light scattering studies of hyaluronic acid solutions.

Abstract Autocorrelation functions for the intensity of laser light scattered from solutions of hyaluronic acid have been measured under a variety of experimental conditions. The form of these functions is consistent with the large amount of long-range intra- and intermolecular interactions characteristic of polyelectrolyte solutions. Further experiments have been performed in order to study the effect of hyaluronic acid on the Brownian diffusion of polystyrene spheres. Using several different sphere sizes as solution probes, the importance of the ionic environment in determining hyaluronic confirmation has been demonstrated.

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.

Relationship between transmembrane potential and activation of motility in rainbow trout (Salmo gairdneri).

A hypothesis is developed that activation of motility in rainbow trout spermatozoa is a result of membrane hyperpolarization. This hypothesis was developed to explain experimental observations of a relationship between membrane potential and motility as revealed by the use of voltage sensitive fluorescent dyes. The results lead to the following conclusions: a) Transmembrane potential hyperpolarizes with decreasing KCl concentration in 100 mM NaCl. b) Transmembrane potential hyperpolarizes with decreasing NaCl concentration. c) NaCl is three time less effective in changing transmembrane potential and two orders of magnitude less effective in inhibiting activation of motility than KCl. d) Chloride ions have little effect on transmembrane potential or motility. e) Increases in osmotic pressure with the non-ionic molecule sucrose increased the amount of KCl required to inhibit activation. f) The major effect of Na+ on K+ inhibition may be osmotic.It is suggested that while sperm cells are in the seminal plasma in the reproductive tract of the male rainbow trout their transmembrane potential is maintained above an activation threshold, probably through Na/K pumps which are found in almost all animal cells. Since K+ is the most important ion in determining the transmembrane potential, hyperpolarization of the plasma membrane below an activation threshold occurs when the sperm cells are diluted, during spawning, into the low K+ environment of freshwater.