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S H White

Publications and source records attributed to S H White.

At least 55 records · Page 3Linked to original sources
PubMed1997

Critical role of lipid composition in membrane permeabilization by rabbit neutrophil defensins.

We have examined the interactions of the six known rabbit neutrophil defensin antimicrobial peptides with large unilamellar vesicles (LUV) made from various lipid mixtures based on the lipid composition of Escherichia coli membranes. We find that the permeabilization of LUV made from E. coli whole lipid extracts differs dramatically from that of single-component LUV made from palmitoyl-oleoyl-phosphatidylglycerol (POPG). Specifically, defensins NP-1, NP-2, NP-3A, NP-3B, and a natural mixture of the six defensins cause fast nonpreferential leakage of high molecular weight dextrans as well as the low molecular weight fluorophore/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) from E. coli whole lipid LUV through large, transient membrane lesions. In contrast, release of ANTS/DPX from POPG LUV induced by the defensins is slow and graded with preference for DPX (Hristova, K., Selsted, M. E., and White, S. H. (1996) Biochemistry 35, 11888-11894). Interestingly, defensins NP-4 and NP-5 alone do not induce leakage from E. coli whole lipid LUV, whereas only NP-4 is ineffective with POPG LUV. Examination of the sequences of the six defensins suggests that the inactivity of NP-4 and NP-5 may be due to their lower net positive charge and/or the substitution of a Thr for the Arg or Lys that follows the fourth Cys residue. We found the presence of three major lipid components of E. coli whole lipid to be essential for creation of the large lesions observed in LUV: phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Cardiolipin appears to play a key role because no leakage can be induced when only phosphatidylglycerol and phosphatidylethanolamine are present. These results indicate the importance of membrane lipid composition in the permeabilization of cell membranes by rabbit defensins.

Amino Acid Sequence↗
PubMed1997

Bilayer interactions of indolicidin, a small antimicrobial peptide rich in tryptophan, proline, and basic amino acids.

Tryptophan, proline, and basic amino acids have all been implicated as being important in the assembly and structure of membrane proteins. Indolicidin, an antimicrobial 13-residue peptide-amide isolated from the cytoplasmic granules of bovine neutrophils, is highly enriched in these amino acids: five tryptophans, three prolines, three basic residues, and no acidic residues. Consistent with the likely importance of these amino acids in membrane protein assembly, indolicidin is known to be highly membrane-active and is believed to act by disruption of cell membranes. We have, therefore, examined the interactions of native indolicidin with large unilamellar vesicles (LUV) formed from palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylglycerol (POPG), in order to use it as a model system for studying membrane protein insertion and for evaluating the relative contributions of hydrophobic and electrostatic forces in peptide-bilayer interactions. Equilibrium dialysis measurements indicate that indolicidin binds strongly, but reversibly, to both neutral POPC and anionic POPG vesicles with free energies of transfer of -8.8 +/- 0.2 and -11.5 +/- 0.4 kcal/mol, respectively. The extremely strong partitioning into POPG vesicles necessitated the development of a new equilibrium dialysis method that is described in detail. Tryptophan fluorescence measurements show that indolicidin is located in the bilayer interface and that indole fluorescence is affected by the type of lipid used to form the LUVs. Circular dichroism (CD) measurements reveal unordered conformations in aqueous and bulk organic solutions and a somewhat more ordered, but not alpha-helical, conformation in SDS micelles and lipid bilayers. Fluorescence requenching measurements (Ladokhin et al. 1995. Biophys. J. 69:1964-1971) on vesicles loaded with the fluorophore/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX), show that indolicidin induces membrane permeabilization. For anionic POPG, leakage is graded with a high preference for the release of cationic DPX over anionic ANTS. For neutral POPC vesicles no such preference is observed. Leakage induction is more effective with POPG vesicles than with POPC vesicles, as judged by three quantitative measures that are developed in the Appendix.

Animals↗
PubMed1997

Sizing membrane pores in lipid vesicles by leakage of co-encapsulated markers: pore formation by melittin.

Many toxins and antimicrobial peptides permeabilize membrane vesicles by forming multimeric pores. Determination of the size of such pores is an important first step for understanding their structure and the mechanism of their self-assembly. We report a simple method for sizing pores in vesicles based on the differential release of co-encapsulated fluorescently labeled dextran markers of two different sizes. The method was tested using the bee venom peptide melittin, which was found to form pores of 25-30 A diameter in palmitoyloleoylphosphatidylcholine (POPC) vesicles at a lipid-to-peptide ratio of 50. This result is consistent with observations on melittin pore formation in erythrocytes (Katsu, T., C. Ninomiya, M. Kuroko, H. Kobayashi, T. Hirota, and Y. Fujita 1988. Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane Biochim. Biophys. Acta. 939:57-63).

Chromatography, Gel↗
PubMed1996

Interactions of monomeric rabbit neutrophil defensins with bilayers: comparison with dimeric human defensin HNP-2.

Human antimicrobial neutrophil defensin HNP-2 has been shown to form large multimeric pores in pure 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG) bilayers that lead to all-or-none release of vesicle contents [Wimley et al. (1994) Protein Sci.3, 1362-1373]. Because human neutrophil defensins form natural dimers in solution, the question arises as to the role of dimerization in pore formation. However, the dimers are so stable that this question is not easily answered directly. Rabbit neutrophil defensins, whose three-dimensional structures are very similar to those of human defensins, are monomeric in aqueous solution and thus provide an opportunity to test the hypothesis that dimerization may play a role in multimeric pore formation. We therefore examined the interactions of the six known rabbit neutrophil defensins with large unilamellar vesicles (LUV) under the conditions known to lead to stable pore formation by HNP-2. We find that the rabbit defensins bind strongly to LUVs formed from pure POPG or mixtures of POPG with neutral (zwitterionic) phospholipid but induce leakage of vesicle contents only from pure POPG vesicles. Rabbit defensin NP-4 does not cause leakage under any conditions examined. The remaining defensins, NP-1, NP-2, NP-3A, NP-3B, and NP-5, cause graded release of the contents of pure POPG vesicles as does a mixture of the six defensins. The graded release indicates that the rabbit defensins do not form stable pores in the membrane. This result thus suggests that the structural features of human defensins that permit dimer formation in aqueous solution are likely to be important in the formation of multimeric pores.

Amino Acid Sequence↗
PubMed1996

Solvation energies of amino acid side chains and backbone in a family of host-guest pentapeptides.

Octanol-to-water solvation free energies of acetyl amino amides (Ac-X-amides) [Fauchère, J.L., & Pliska, V. (1983) Eur. J. Med. Chem. --Chim. Ther. 18,369] form the basis for computational comparisons of protein stabilities by means of the atomic solvation parameter formalism of Eisenberg and McLachlan [(1986) Nature 319, 199]. In order to explore this approach for more complex systems, we have determined by octanol-to-water partitioning the solvation energies of (1) the guest (X) side chains in the host-guest pentapeptides AcWL-X-LL, (2) the carboxy terminus of the pentapeptides, and (3) the peptide bonds of the homologous series of peptides AcWLm (m = 1-6). Solvation parameters were derived from the solvation energies using estimates of the solvent-accessible surface areas (ASA) obtained from hard-sphere Monte Carlo simulations. The measurements lead to a side chain solvation-energy scale for the pentapeptides and suggest the need for modifying the Asp, Glu, and Cys values of the "Fauchère-Pliska" solvation-energy scale fro the Ac-X-amides. We find that the unfavorable solvation energy of nonpolar residues can be calculated accurately by a solvation parameter of 22.8 +/- 0.8 cal/mol/A2, which agrees satisfactorily with the AC-X-amide data and thereby validates the Monte Carlo ASA results. Unlike the Ac-X-amide data, the apparent solvation energies of the uncharged polar residues are also largely unfavorable. This unexpected finding probably results, primarily, from differences in conformation and hydrogen bonding in octanol and buffer but may also be due to the additional flaking peptide bonds of the pentapeptides. The atomic solvation parameter (ASP) for the peptide bond is comparable to the ASP of the charged carboxy terminus which is an order of magnitude larger than the ASP of the uncharged polar side chains of the Ac-X-amides. The very large peptide bond ASP, -96 +/- 6 cal/mol/A2, profoundly affects the results of computational comparisons of protein stability which use ASPs derived from octanol-water partitioning data.

Amides↗
PubMed1996

Direct measurement of salt-bridge solvation energies using a peptide model system: implications for protein stability.

The solvation energies of salt bridges formed between the terminal carboxyl of the host pentapeptide AcWL- X-LL and the side chains of Arg or Lys in the guest (X) position have been measured. The energies were derived from octanol-to-buffer transfer free energies determined between pH 1 and pH 9. 13C NMR measurements show that the salt bridges form in the octanol phase, but not in the buffer phase, when the side chains and the terminal carboxyl group are charged. The free energy of salt-bridge formation in octanol is approximately -4 kcal/mol (1 cal = 4.184 J), which is equal to or slightly larger than the sum of the solvation energies of noninteracting pairs of charged side chains. This is about one-half the free energy that would result from replacing a charge pair in octanol with a pair of hydrophobic residues of moderate size. Therefore, salt bridging in octanol can change the favorable aqueous solvation energy of a pair of oppositely charged residues to neutral or slightly unfavorable but cannot provide the same free energy decrease as hydrophobic residues. This is consistent with recent computational and experimental studies of protein stability.

Amino Acid Sequence↗
PubMed1996

Experimentally determined hydrophobicity scale for proteins at membrane interfaces.

The partitioning of membrane-active oligopeptides into membrane interfaces promotes the formation of secondary structure. A quantitative description of the coupling of structure formation to partitioning, which may provide a basis for understanding membrane protein folding and insertion, requires an appropriate free energy scale for partitioning. A complete interfacial hydrophobicity scale that includes the contribution of the peptide bond was therefore determined from the partitioning of two series of small model peptides into the interfaces of neutral (zwitterionic) phospholipid membranes. Aromatic residues are found to be especially favoured at the interface while charged residues, and the peptide bond, are disfavoured about equally. Reduction of the high cost of partitioning the peptide bond through hydrogen bonding may be important in the promotion of structure formation in the membrane interface.

Animals↗
PubMed1995

Structure, function, and membrane integration of defensins.

Defensins comprise a structural class of small cationic peptides that exert broad-spectrum antimicrobial activities through membrane permeabilization. Their predominantly beta-sheet structure, stabilized by three disulfide bonds, distinguishes them from other antimicrobial peptides which typically form amphiphilic helices. Defensins bind to membranes electrostatically and subsequently form apparently multimeric pores. Recent structural and biophysical studies are beginning to provide insights into the process of permeabilization.

Amino Acid Sequence↗
PubMed1995

Leakage of membrane vesicle contents: determination of mechanism using fluorescence requenching.

Agents such as antimicrobial peptides and toxins can permeabilize membrane vesicles to cause leakage of entrapped contents in either a graded or an all-or-none fashion. Determination of which mode of leakage is induced is an important step in understanding the molecular mechanism of membrane permeabilization. Wimley et al. (1994, Protein Sci. 3:1362-1378) have developed a fluorescence method for distinguishing the two modes that makes use of the dye/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) without the usual need for the physical separation of vesicles from released contents. Their "requenching" method establishes the mode of release through the fluorescence changes that occur when DPX is added externally to a solution of vesicles that have released some fraction of their contents. However, the requenching method as originally stated ignored the possibility of preferential release of dye or quencher. Here we extend the theory of the method to take into account preferential release and the effects of graded leakage. The ratio of the rates of release of the cationic quencher DPX and anionic dye 8-aminonapthalene-1,3,6 trisulfonic acid can be estimated by means of the theory. For graded leakage, we show that the release of the markers does not coincide with the fluorescence changes observed in the standard leakage assay. This is true for self-quenching dyes as well and means that 1) the amount of released material will be overestimated and 2) the kinetics will be nonexponential and have artificially high apparent rates. We show how the extended requenching analysis allows the results of leakage experiments to be corrected for artifacts that result from graded and preferential leakage. Experimental evidence is presented for the existence of peptide-induced preferential graded leakage of DPX from both neutral and anionic vesicles.

Amino Acid Sequence↗
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↗
PubMed1994

The evolution of proteins from random amino acid sequences: II. Evidence from the statistical distributions of the lengths of modern protein sequences.

This paper continues an examination of the hypothesis that modern proteins evolved from random heteropeptide sequences. In support of the hypothesis, White and Jacobs (1993, J Mol Evol 36:79-95) have shown that any sequence chosen randomly from a large collection of nonhomologous proteins has a 90% or better chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. The goal of the present study was to investigate the possibility that the random-origin hypothesis could explain the lengths of modern protein sequences without invoking specific mechanisms such as gene duplication or exon splicing. The sets of sequences examined were taken from the 1989 PIR database and consisted of 1,792 "super-family" proteins selected to have little sequence identity, 623 E. coli sequences, and 398 human sequences. The length distributions of the proteins could be described with high significance by either of two closely related probability density functions: The gamma distribution with parameter 2 or the distribution for the sum of two exponential random independent variables. A simple theory for the distributions was developed which assumes that (1) protoprotein sequences had exponentially distributed random independent lengths, (2) the length dependence of protein stability determined which of these protoproteins could fold into compact primitive proteins and thereby attain the potential for biochemical activity, (3) the useful protein sequences were preserved by the primitive genome, and (4) the resulting distribution of sequence lengths is reflected by modern proteins. The theory successfully predicts the two observed distributions which can be distinguished by the functional form of the dependence of protein stability on length. The theory leads to three interesting conclusions. First, it predicts that a tetra-nucleotide was the signal for primitive translation termination. This prediction is entirely consistent with the observations of Brown et al. (1990a,b, Nucleic Acids Res 18:2079-2086 and 18: 6339-6345) which show that tetra-nucleotides (stop codon plus following nucleotide) are the actual signals for termination of translation in both prokaryotes and eukaryotes. Second, the strong dependence of statistical length distributions on sequence-termination signaling codes implies that the evolution of stop codons and translation-termination processes was as important as gene splicing in early evolution. Third, because the theory is based upon a simple no-exon stochastic model, it provides a plausible alternative to a limited universe of exons from which all proteins evolved by gene duplication and exon splicing (Dorit et al. 1990, Science 250:1377-1382).

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↗