The effect of hydrogen bonding on the diffusion of water in n-alkanes and n-alcohols measured with a novel single microdroplet method.

While the Stokes-Einstein (SE) equation predicts that the diffusion coefficient of a solute will be inversely proportional to the viscosity of the solvent, this relation is commonly known to fail for solutes, which are the same size or smaller than the solvent. Multiple researchers have reported that for small solutes, the diffusion coefficient is inversely proportional to the viscosity to a fractional power, and that solutes actually diffuse faster than SE predicts. For other solvent systems, attractive solute-solvent interactions, such as hydrogen bonding, are known to retard the diffusion of a solute. Some researchers have interpreted the slower diffusion due to hydrogen bonding as resulting from the effective diffusion of a larger complex of a solute and solvent molecules. We have developed and used a novel micropipette technique, which can form and hold a single microdroplet of water while it dissolves in a diffusion controlled environment into the solvent. This method has been used to examine the diffusion of water in both n-alkanes and n-alcohols. It was found that the polar solute water, diffusing in a solvent with which it cannot hydrogen bond, closely resembles small nonpolar solutes such as xenon and krypton diffusing in n-alkanes, with diffusion coefficients ranging from 12.5x10(-5) cm(2)/s for water in n-pentane to 1.15x10(-5) cm(2)/s for water in hexadecane. Diffusion coefficients were found to be inversely proportional to viscosity to a fractional power, and diffusion coefficients were faster than SE predicts. For water diffusing in a solvent (n-alcohols) with which it can hydrogen bond, diffusion coefficient values ranged from 1.75x10(-5) cm(2)/s in n-methanol to 0.364x10(-5) cm(2)/s in n-octanol, and diffusion was slower than an alkane of corresponding viscosity. We find no evidence for solute-solvent complex diffusion. Rather, it is possible that the small solute water may be retarded by relatively longer residence times (compared to non-H-bonding solvents) as it moves through the liquid.

Amyloid-type fiber formation in control of enzyme action: interfacial activation of phospholipase A2.

The lag-burst behavior in the action of phospholipase A(2) (PLA(2)) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature T(m) of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Förster resonance energy transfer between donor- and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA(2), evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA(2), involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form "mature" fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis.

A versatile method for determining the molar ligand-membrane partition coefficient.

A novel method for the quantitative assessment of the membrane partitioning of a ligand from the aqueous phase is described, demonstrated here with the thoroughly studied antipsychotic chlorpromazine (CPZ). More specifically, collisional quenching of the fluorescence of a pyrene labeled fluorescent lipid analog 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by CPZ was utilized, using 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and -serine (POPC and POPS) liposomes as model membranes. The molar partition coefficient is obtained from two series of titrations, one with constant [phospholipid] and increasing [drug] and the other with constant [drug] and varying total [phospholipid], the latter further comprising of large unilamellar vesicles (LUVs) of POPC/POPS/PPDPC at a constant concentration of 10 microM and indicated concentrations of POPC/POPS LUVs. Notably, the approach described is generic and can be employed in screening for the membrane partitioning of compounds, providing that a suitable fluorescence parameter can be incorporated into one population of liposomes utilized as model membranes.

Characteristics of fibers formed by cytochrome c and induced by anionic phospholipids.

Recent publications described the formation of millimeter-length fibers by diverse lipid-binding proteins (e.g., histone H1, cytochrome c, indolicidin, and endostatin) when they are mixed with 80:20 phosphatidylcholine/phosphatidylserine vesicles. Further, these fibers displayed amyloid characteristics when stained with Congo Red. In the study presented here, we found by FTIR the amide I absorption band to reveal significant variation in fibers formed by cytochrome c, with some consisting of cytochrome c in a nativelike conformation and some exhibiting strong amyloid (beta-sheet) characteristics. Protein structure also varied from amyloid to nearly native within single fibers. Fibers were frequently blue or bluish and sometimes iridescent, likely due to interference of light in the fibers. The amyloid-type amide I band was observed for blue fibers only. AFM shows that fibers consist of smaller 3-4 nm diameter fibers with 10 nm lateral spacing.

Mutational analysis and membrane-interactions of the beta-sheet-like N-terminal domain of the pediocin-like antimicrobial peptide sakacin P.

To gain insight into how the N-terminal three-stranded beta-sheet-like domain in pediocin-like antimicrobial peptides positions itself on membranes, residues in the well-conserved (Y)YGNGV-motif in the domain were substituted and the effect of the substitutions on antimicrobial activity and binding of peptides to liposomes was determined. Peptide-liposome interactions were detected by measuring tryptophan-fluorescence upon exposing liposomes to peptides in which a tryptophan residue had been introduced in the N-terminal domain. The results revealed that the N-terminal domain associates readily with anionic liposomes, but not with neutral liposomes. The electrostatic interactions between peptides and liposomes facilitated the penetration of some of the peptide residues into the liposomes. Measuring the antimicrobial activity of the mutated peptides revealed that the Tyr2Leu and Tyr3Leu mutations resulted in about a 10-fold reduction in activity, whereas the Tyr2Trp, Tyr2Phe, Tyr3Trp and Tyr3Phe mutations were tolerated fairly well, especially the mutations in position 3. The Val7Ile mutation did not have a marked detrimental effect on the activity. The Gly6Ala mutation was highly detrimental, consistent with Gly6 being in one of the turns in the beta-sheet-like N-terminal domain, whereas the Gly4Ala mutation was tolerated fairly well. All mutations involving Asn5, including the conservative mutations Asn5Gln and Asn5Asp, were very deleterious. Thus, both the polar amide group on the side chain of Asn5 and its exact position in space were crucial for the peptides to be fully active. Taken together, the results are consistent with Val7 positioning itself in the hydrophobic core of target membranes, thus forcing most of the other residues in the N-terminal domain into the membrane interface region: Tyr3 and Asn5 in the lower half with their side chains pointing downward and approaching the hydrophobic core, Tyr2, Gly4 and His8 and 12 in the upper half, Lys1 near the middle of the interface region, and the side chain of Lys11 pointing out toward the membrane surface.

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: implications for a novel mechanism of action.

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.

Binding of endostatin to phosphatidylserine-containing membranes and formation of amyloid-like fibers.

Endostatin, the 20-kDa C-terminal NC1 domain of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis and tumor growth. A major problem in reconciling the many reported in vitro effects of endostatin is the lack of a high-affinity receptor, and a search for the latter continues. In accordance with the above, the molecular mechanisms of action of endostatin remain elusive. We show here that endostatin binds to membranes containing acidic phospholipids, phosphatidylserine (PS) or phosphatidylglycerol (PG). More specifically, a red shift in the fluorescence emission of Trp of endostatin in the presence of liposomes containing these anionic lipids was evident, revealing the average environment of Trps to become less hydrophobic. This shift was not observed for phosphatidylcholine (PC) liposomes, demonstrating the acidic lipid to be required. Quenching by endostatin of the fluorescence of a pyrene-labeled phospholipid analogue in PS containing membranes was seen, while there was no effect for PC liposomes. Resonance energy transfer from the Trp residues of endostatin to a dansyl-labeled phospholipid further confirmed the association of endostatin with PS-containing membranes, whereas there was no binding to PC liposomes. Intriguingly, the association of endostatin with PS-containing liposomes triggered the formation of fibers, with Congo red staining producing green birefringence characteristic for amyloid. Lipid was incorporated into these fibers, as shown by staining when a trace amount (X = 0.02) of fluorescent phospholipid analogues was present in the liposomes. No fiber formation was seen when endostatin was added to liposomes composed of PC only. Because PS has been reported to be exposed in the outer surface of the plasma membrane of cancer cells and vascular endothelial cells, our results suggest that this lipid could represent a target for endostatin in the cancer cell surface and tumors, thus suggesting a novel mechanism of its action. More specifically, analogous to a number of other cytotoxic proteins interacting with negatively charged lipids, PS-triggered fiber formation by endostatin on the surface of cancer cells would impair the permeability barrier function of the plasma membrane, resulting in cell death.

Fluorescence spectroscopic characterization of Humicola lanuginosa lipase dissolved in its substrate.

The conformational dynamics of Humicola lanuginosa lipases (HLL) and its three mutants were investigated by steady state and time-resolved fluorescence spectroscopy in two different media, aqueous buffer and the substrate triacetin. The fluorescence of the four Trps of the wild-type HLL (wt) reports on the global changes of the whole lipase molecule. In order to monitor conformational changes specifically in the alpha-helical surface loop, the so-called 'lid' of HLL comprised of residues 86-93, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Mutants W89L and W89mN33Q (W117F, W221H, W260H, N33Q) were used to survey the impact of Trp89 and mannose residues, respectively. Based on the data obtained, the following conclusions can be drawn. (i) HLL adapts the 'open' conformation in triacetin, with the alpha-helical surface loop moving so as to expose the active site. (ii) Trp89 contained in the lid plays an unprecedently important role in the structural stability of HLL. (iii) In triacetin, but not in the buffer, the motion of the Trp89 side chain becomes distinguishable from the motion of the lid. (iv) The carbohydrate moiety at Asn33 has only minor effects on the dynamics of Trp89 in the lid as judged from the fluorescence characteristics of the latter residue.

Evidence for the lack of a specific interaction between cholesterol and sphingomyelin.

The putative specific interaction and complex formation by sphingomyelin and cholesterol was investigated. Accordingly, low contents (1 mol % each) of fluorescently labeled derivatives of these lipids, namely 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PyrPC), n-[10-(1-pyrenyl)decanoyl]sphingomyelin (PyrSM), and increasing concentrations of cholesterol (up to 5 mol %), were included in large unilamellar vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC), and the excimer/monomer fluorescence emission ratio (I(e)/I(m)) was measured. In DNPC below the main phase transition, the addition of up to 5 mol % cholesterol reduced I(e)/I(m) significantly. Except for this, cholesterol had only a negligible effect in both matrices and for both probes. We then compared the efficiency of resonance energy transfer from PyrPC and PyrSM to 22-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBDchol). An augmenting colocalization of the latter resonance energy transfer pair with temperature was observed in a DMPC matrix below the main phase transition. In contrast, compared to PyrSM the colocalization of PyrPC with NBDchol was more efficient in the longer DNPC matrix. These results could be confirmed using 5,6-dibromo-cholestan-3beta-ol as a collisional quencher for the pyrene-labeled lipids. The results indicate lack of a specific interaction between sphingomyelin and cholesterol, and further imply that hydrophobic mismatch between the lipid constituents could provide the driving force for the cosegregation of sphingomyelin and cholesterol in fluid phospholipid bilayers of thicknesses comparable to those found for biomembranes.