Biomedical subjects

W C Wimley

Publications and source records attributed to W C Wimley.

25 records · Page 2Linked to original sources

PubMed1994

Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores.

Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 microM POPG) readily aggregated at HNP-2 concentrations above 1 microM, but no mixing of vesicle contents could be detected for concentrations as high as 2 microM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 microM POPG vesicles, 50% of entrapped solute is released by 0.7 microM HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (approximately 400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.

Amino Acid Sequence↗

PubMed1993

Membrane partitioning: distinguishing bilayer effects from the hydrophobic effect.

The free energy of transfer of nonpolar solutes from water to lipid bilayers is often dominated by a large negative enthalpy rather than the large positive entropy expected from the hydrophobic effect. This common observation has led to the concept of the "nonclassical" hydrophobic effect and the idea that the "classical" hydrophobic effect may not drive partitioning in many bilayer systems. We show through measurements of the heat capacity changes associated with the partitioning of tryptophan side-chain analogs into lipid bilayers and into bulk cyclohexane that the hydrophobic effect plays a crucial role regardless of the large negative enthalpy. The results emphasize that bulk-phase measurements are inadequate for describing bilayer partitioning. The experimental approach described should be generally useful for analyzing the bilayer interactions of a broad range of biologically important molecules.

Chromatography, High Pressure Liquid↗

PubMed1993

Quantitation of electrostatic and hydrophobic membrane interactions by equilibrium dialysis and reverse-phase HPLC.

Equilibrium dialysis and reverse-phase HPLC have been used for the sensitive and precise quantitation of both electrostatic and hydrophobic interactions of peptides and small molecules with lipid bilayers. We show that hydrophobic solutes are rapidly and quantitatively released from lipid dispersions when loaded onto a C4 reverse-phase HPLC column equilibrated in water+0.1% trifluoroacetic acid and that the lipid molecules have no interfering effect on the chromatography. Peptides interacting electrostatically with bilayers are released quantitatively when a higher ionic strength buffer (water+2% ammonium acetate) is used. As little as 50 ng of solute can be accurately quantitated even in the presence of milligram amounts of lipid. We demonstrate the application of these methods to the hydrophobic interactions between indoles and lipid bilayers and to the electrostatic interaction between defensins, which are cationic antibiotic peptides, and anionic bilayers. The high sensitivity allows nondestructive quantitation of submicrogram amounts of precious solutes and the high precision allows the heat capacity change, an important thermodynamic parameter, to be obtained from the partitioning data.

Antifungal Agents↗

PubMed1992

Partitioning of tryptophan side-chain analogs between water and cyclohexane.

We have measured the partitioning of the tryptophan side-chain analogs 3-methylindole and N-methylindole between water and cyclohexane over the temperature range 8-55 degrees C to investigate the relative contribution of the imine-NH- to the free energy of transfer. We take advantage of the fact that the indole imine nitrogen is blocked by a methyl group in N-methylindole. Unlike previous studies, we take into account the water present in the cyclohexane phase. Free energies of partitioning were calculated using mole-fraction, volume-fraction, and Flory-Huggins-corrected volume-fraction partition coefficients [De Young, L. R., & Dill, K. A. (1990) J. Phys. Chem. 94, 801-809; Sharp, K. A., Nicholls, A., Friedman, R., & Honig, B. (1991) Biochemistry 30, 9686-9697]. These approaches account for configurational entropy changes in different ways and thus lead to different values for the calculated free energies of transfer. There is a 2-3-fold difference in the free energies calculated from our measurements, using the different units. Independent of units, the partitioning of both compounds involves identical entropy changes. However, 3-methylindole has an additional unfavorable enthalpic contribution to partitioning into cyclohexane of +1.6 kcal/mol (independent of units) which is presumably the cost of removing the indole -NH- group from water and transferring it to cyclohexane. In cyclohexane, 3-methylindole forms hydrogen bonds with water that cause water to copartition into cyclohexane with the solute. A method is described which allows the partitioning process to be examined independent of subsequent interactions with water in the solvent.

Chemical Phenomena↗

PubMed1991

Phosphatidylethanolamine enhances the concentration-dependent exchange of phospholipids between bilayers.

It has previously been demonstrated that lipid exchange between phosphatidylcholine vesicles, at higher concentrations, is characterized by a second-order concentration-dependent exchange process in addition to the first-order process operative at lower concentrations (Jones, J. D., & Thompson, T. E. (1989) Biochemistry 28, 129-134). Furthermore, it was demonstrated that the second-order process occurs as a result of an enhancement of the first-order desorption process, possibly resulting from attractive interactions between a potentially desorbing lipid molecule and a transiently apposed bilayer (Jones, J. D., & Thompson, T. E. (1990) Biochemistry 29, 1593-1600). In this work we have studied the exchange of [3H]dimyristoylphosphatidylcholine (DMPC) between large vesicles of the compositions 100% DMPC, 70/30 (mol/mol) DMPC/dimyristoylphosphatidylethanolamine (DMPE), and 68.25/30/1.75 (mol/mol/mol) DMPC/DMPE/dimyristoylphosphatidylglycerol (DMPG). The second-order exchange process is enhanced by 100-fold or more in vesicles containing 30 mol % DMPE relative to 100% DMPC and is reduced or eliminated by the addition of 1.75% of the anionic lipid DMPG. These effects can be achieved by alterations in the equilibrium bilayer separation of 5 A or less. The results are in accord with the model of Jones and Thompson and indicate that relatively low concentrations of PE in a PC bilayer can have significant effects on bilayer surface properties and on potential interactions between bilayers.

Biological Transport↗

PubMed1991

Transbilayer and interbilayer phospholipid exchange in dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine large unilamellar vesicles.

The rates of spontaneous interbilayer and transbilayer exchange of [3H]dimyristoylphosphatidylcholine ([3H]DMPC) were examined in DMPC and DMPC/dimyristoylphosphatidylethanolamine (DMPE) large unilamellar vesicles in the liquid-crystalline-, gel-, and mixed-phase states. DMPC desorption rates from either gel or liquid-crystalline phases containing DMPE are very similar to the corresponding rates from pure DMPC gel or liquid-crystalline phases. This is not the case for DMPC desorption from distearoylphosphatidylcholine (DSPC)-containing gel phases, where the desorption rates are significantly faster than from a pure DMPC gel phase [Wimley, W. C., & Thompson, T. E. (1990) Biochemistry 29, 1296-1303]. We proposed that the DMPC/DSPC behavior results from packing defects in gel phases composed of both DMPC and DSPC molecules because of the four-carbon difference in the acyl chain lengths of the two species. The present results strongly support this hypothesis because no such anomalous behavior is observed in DMPC/DMPE, which is similar to DMPC/DSPC in phase behavior but does not have the chain length difference. The inclusion of 10-30 mol % DMPE in DMPC bilayers was also found to have a significant effect on the rate of transbilayer movement (flip-flop) of [3H]DMPC in the liquid-crystalline phase. Between 10 and 30 mol % DMPE, flip-flop of DMPC is slowed by at least 10-fold relative to flip-flop in DMPC bilayers, and the entropy and enthalpy of flip-flop activation are both substantially decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Radioisotopes↗

PubMed1990

Exchange and flip-flop of dimyristoylphosphatidylcholine in liquid-crystalline, gel, and two-component, two-phase large unilamellar vesicles.

The rate and extent of spontaneous exchange of dimyristoylphosphatidylcholine (DMPC) from large unilamellar vesicles (LUV) composed of either DMPC or mixtures of DMPC/distearoylphosphatidylcholine (DSPC) have been examined under equilibrium conditions. The phase state of the vesicles ranged from all-liquid-crystalline through mixed gel/liquid-crystalline to all-gel. The exchange rate of DMPC between liquid-crystalline DMPC LUV, measured between 25 and 55 degrees C, was found to have an Arrhenius activation energy of 24.9 +/- 1.4 kcal/mol. This activation energy and the exchange rates are very similar to those obtained for the exchange of DMPC between DMPC small unilamellar vesicles (SUV). The extent of exchange of DMPC in LUV was found to be approximately 90%. This is in direct contrast to the situation in DMPC SUV where only the lipid in the outer monolayer is available for exchange. Thus, transbilayer movement (flip-flop) is substantially faster in liquid-crystalline DMPC LUV than in SUV. Desorption from gel-phase LUV has a much lower rate than gel-phase SUV with an activation energy of 31.7 +/- 3.7 kcal/mol compared to 11.5 +/- 2 kcal/mol reported for SUV. A defect-mediated exchange in gel-phase SUV, which is not the major pathway for exchange in LUV, is proposed on the basis of the thermodynamic parameters of the activation process. Surprisingly, the rates of DMPC exchange between DMPC/DSPC two-component LUV, measured over a wide range of compositions and temperatures, were found to exhibit very little dependence on the composition or phase configuration of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallins↗