Pekka Mustonen

Lipid dynamics and peripheral interactions of proteins with membrane surfaces

A large body of evidence strongly indicates biomembranes to be organized into compositionally and functionally specialized domains, supramolecular assemblies, existing on different time and length scales. For these domains and intimate coupling between their chemical composition, physical state, organization, and functions has been postulated. One important constituent of biomembranes are peripheral proteins whose activity can be controlled by non-covalent binding to lipids. Importantly, the physical chemistry of the lipid interface allows for a rapid and reversible control of peripheral interactions. In this review examples are provided on how membrane lipid (i) composition (i.e., specific lipid structures), (ii) organization, and (iii) physical state can each regulate peripheral binding of proteins to the lipid surface. In addition, a novel and efficient mechanism for the control of the lipid surface association of peripheral proteins by [Ca2+], lipid composition, and phase state is proposed. The phase state is, in turn, also dependent on factors such as temperature, lateral packing, presence of ions, metabolites and drugs. Confining reactions to interfaces allows for facile and cooperative large scale integration and control of metabolic pathways due to mechanisms which are not possible in bulk systems.

Differential scanning calorimetry study on the binding of nucleic acids to dimyristoylphosphatidylcholine-sphingosine liposomes

Binding of DNA and RNA to sphingosine-containing dimyristoylphosphatidylcholine (DMPC) liposomes was characterized by differential scanning calorimetry. The thermal phase behaviour of neat DMPC liposomes was unaffected by the presence of the nucleic acids. However, significant alterations in the melting profiles of the DMPC/sphingosine composite membranes were produced by DNA and RNA, thus revealing their binding to the liposomes. For example, for 79:21 (molar ratio) DMPC/sphingosine liposomes a single endotherm at 29.1 degrees C with an enthalpy of 6.3 kcal/mol lipid was observed. In the presence of DNA at the nucleotide/sphingosine ratio of 0.6 this endotherm separated into three distinct peaks at 28.0, 31.4 and 35.1 degrees C, together with an approximately 22% reduction in the total enthalpy. Further increase in DNA concentration up to 1.5 nucleotides per sphingosine led to complete loss of the original heat absorption peak of the DMPC/sphingosine liposomes, while an endotherm at 34.3 degrees C with delta H of 2.7 kcal/mol developed. By visual inspection, rapid and extensive aggregation of the liposomes due to DNA was evident. Evidence for DNA-induced phase separation was also provided by compression isotherms of sphingosine containing DMPC monolayers recorded over an aqueous buffer both in the presence and absence of DNA. The effects of RNA on the thermal phase behaviour of the composite liposomes were qualitatively similar to those described above for DNA. Notably, the presence of eggPA abolished the nucleic acid induced heat capacity changes for DMPC/sphingosine liposomes probably because of neutralization of the positive charge of sphingosine. The binding of DNA to DMPC/sphingosine liposomes occurred both below and above the lipid phase transition temperature, as shown by fluorescence resonance energy transfer utilizing adriamycin-labelled DNA as a quencher and membrane incorporated pyrene-labelled phospholipid as a donor. However, the apparent binding to liquid crystalline liposomes was slightly more effective.

Sphingosine-mediated membrane association of DNA and its reversal by phosphatidic acid

Resonance energy transfer was measured between egg phosphatidylcholine liposomes containing the intramolecular excimer forming pyrene-labelled phospholipid analogue 1,2-bis[pyren-1-(-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC) as a donor and DNA-bound adriamycin as an acceptor. Membrane association of DNA turned out to be critically dependent on the presence of sphingosine in the liposomes. Identical result was obtained by measuring the extent of quenching of the fluorescent DNA-bound dye Hoechst 33258 due to energy transfer to the lipophilic stain Nile Red incorporated in egg phosphatidylcholine liposomes containing varying amounts of sphingosine. The attachment of DNA to sphingosine-containing membranes could be reversed by the further inclusion of the negatively charged phosphatidic acid up to approximately 1:2 PA/sphingosine molar ratio in the liposomes, thus suggesting the involvement of electrostatic interactions. Differential scanning calorimetry measurements confirmed a lack of association between DNA and dimyristoylphosphatidylcholine liposomes. Instead drastic changes were produced by DNA in the heat capacity scans measured for liposomes also incorporating sphingosine. Fluorescence microscopy revealed an extensive aggregation of sphingosine containing pyrene-phosphatidylcholine-labelled egg phosphatidylcholine liposomes in the presence of DNA. Together with other available data on the effects of sphingosine, the present findings suggest that sphingosine could directly alter the chromatin structure. Accordingly, such alterations may contribute to the control of replication and gene expression.

Influence of sphingosine on the thermal phase behaviour of neutral and acidic phospholipid liposomes

The physical state of lipids is known to have pronounced effects on membrane functions. We studied the influence of sphingosine, a modulator of diverse cellular processes on the thermal phase behaviour and molecular packing of neutral and acidic phospholipids. Differential scanning calorimetry of multilamellar liposomes as well as the monolayer technique were employed. Inclusion of sphingosine in diacylphosphatidylcholine liposomes increased their pretransition temperature Tp until at about 10 mol% sphingosine this transition was abolished. For these liposomes a gradual increase in both the temperature Tm and enthalpy delta Hm of the main transition caused by sphingosine was observed. In contrast to diacylphosphatidylcholines, the Tp for dihexadecylphosphatidylcholine was lowered by sphingosine, demonstrating that the latter destabilizes the interdigitated gel phase. Inclusion of sphingosine in dimyristoylphosphatidic acid and dipalmitoylphosphatidylserine liposomes first elevated the Tm without significant changes in delta Hm, while at sphingosine contents > 50 mol% the appearance of complex melting profiles was evident. The transition temperature for the egg yolk phosphatidic acid was shifted from below 0 to 29 degrees C when mixed with sphingosine in a molar ratio of 1:1. Sphingosine also condensed the eggPA monolayers residing on an air-buffer interface. Accordingly, besides introducing a positive surface charge allowing the binding or activation of some proteins, sphingosine could influence membrane-mediated cellular processes by altering the organization and state of membrane lipids.

Pyrene-Labelled Lipids as Fluorescent Probes in Studies on Biomembranes and Membrane Models

The immense progress in the understanding of the functions of proteins and nucleic acids in living cells is still contrasted by the limited comprehension of the significance of lipids and their structural diversity in the self-assembly and functionalization of biological membranes. Nevertheless, taking into account the costly maintenance of the specific tissue, cell, and cell organelle lipid compositions and their alterations it is obvious that the individual lipid classes do serve more important roles than what is at present commonly accepted [1].

Substrate level modulation of the activity of phospholipase A2 in vitro by 12-O-tetradecanoylphorbol-13-acetate.

The action of porcine pancreatic phospholipase A2 towards fluorescent phospholipid analogs is either enhanced or suppressed by 4 beta-12-O-tetradecanoylphorbol-13- acetate (TPA), depending on the chemical structure of the substrate and the concentration of Ca2+. In the presence of nmolar Ca2+ concentrations increasing [TPA] enhanced by approx. 5-fold the rate of hydrolysis of the pyrene-labelled acidic alkyl-acyl phospholipid, 1-octacosanyl-2-[6- (pyrene-1-yl)] hexanoyl-sn-glycero-3-phosphatidylmethanol. Maximal effect was obtained at high TPA/substrate molar ratios approaching 1:2. In the presence of 4 mM CaCl2 maximal activation was reduced to approximately 1.5-fold. With the corresponding phosphatidylcholine derivative as a substrate increasing [TPA] reduced fatty acid release maximally by 90% both at low [Ca2+] as well as in the presence of 4 mM CaCl2. Essentially identical results were obtained using 4 alpha-TPA, a stereoisomer which does not activate protein kinase C.