Contemporary pollen-mediated gene immigration reflects the historical isolation of a rare, animal-pollinated shrub in a fragmented landscape.

Fragmentation is generally considered to have negative impacts on widespread outbreeders but impacts on gene flow and diversity in patchy, naturally rare, self-compatible plant species remain unclear. We investigated diversity, gene flow and contemporary pollen-mediated gene immigration in the rare, narrowly distributed endemic shrub Calothamnus quadrifidus ssp. teretifolius. This taxon occurs in an internationally recognized biodiversity hotspot subjected to recent human-induced fragmentation and the condition of the remnants ranges from intact to highly degraded. Using microsatellites, we found that inbreeding, historically low gene flow and significant population differentiation have characterized the genetic system of C. quadrifidus ssp. teretifolius. Inbreeding arises from self-pollination, a small amount of biparental inbreeding and significant correlation of outcross paternity but fecundity was high suggesting populations might have purged their lethals. Paternity analyses show that pollinators can move pollen over degraded and intact habitat but populations in both intact and degraded remnants had few pollen parents per seed parent and low pollen immigration. Genetic diversity did not differ significantly between intact and degraded remnants but there were signs of genetic bottlenecks and reduced diversity in some degraded remnants. Overall, our study suggests human-induced fragmentation has not significantly changed the mating system, or pollen immigration to, remnant populations and therefore genetic connectivity need not be the highest conservation priority. Rather, for rare species adapted to higher levels of inbreeding, conservation efforts may be best directed to managing intact habitats and ecosystem processes.

A combined study of aggregation, membrane affinity and pore activity of natural and modified melittin.

The pore activity of melittin and several chemically modified derivatives has been investigated using conductance measurements on planar lipid bilayers and marker release from small unilamellar vesicles. The modifications included N-terminal formylation, acetylation, succinylation and modification of the tryptophan residue. All of the compounds showed bilayer permeabilizing properties, though quantitative differences were evident. These comprised changes in the voltage dependence of the conductance, in the single-pore kinetics, in the concentration of aqueous peptide required to induce a given pore activity and in the apparent 'molecularity' reflected by the power law of its concentration dependence. A strong tendency for disrupting bilayers was not always correlated with strong pore activity. For a better understanding of these results, measurements of pore activity were complemented by studying the aggregation behavior in solution and the water-membrane partition equilibrium. Modifications of charged residues gave rise to significant changes in the aggregation properties, had virtually no influence on the partition coefficient. The latter decreased strongly, however, as a result of tryptophan modification. Analysis of the isotherms was consistent with the assumption that the arginine residues in melittin do not contribute very much to charge accumulation at the immediate membrane/water interface.

Membrane interaction of 'peptide P' derived from the repeating motif of properdin.

A 24 amino acid residue peptide corresponding to the central part of the 'thrombospondin-repeat' motif of the human serum protein properdin was synthesized. The peptide, termed 'peptide P', contains three tryptophans near the N-terminus and an arginine cluster close to the C-terminus. Its sequence closely matches a consensus sequence which has been claimed to characterize a sulfatide binding motif. Membrane binding of peptide P was analyzed using changes in its tryptophan emission upon adding small unilamellar vesicles. The peptide bound to the membranes in a way suggesting simple water/membrane partitioning. Analysis of electrostatic effects at different ionic strengths indicated small electrostatic contributions upon interaction with zwitterionic lipid, despite the large charge number (z = +4) of the peptide. Membrane affinity was increased by one order of magnitude if the bilayers contained 20% of negatively charged lipid. No difference could be detected whether the charged lipid was sulfatide or phosphatidylglycerol. Strong and rapid vesicle aggregation was evident as the peptide associated with the negatively charged vesicles. In addition, a fluorescent energy transfer assay with vesicles and internal total reflection fluorescence microscopy on supported bilayers were used to study membrane interaction of whole human properdin. No sulfatide specificity could be detected.

Surface charging by large multivalent molecules. Extending the standard Gouy-Chapman treatment.

Traditionally, Gouy-Chapman theory has been used to calculate the distribution of ions in the diffuse layer next to a charged surface. In recent years, the same theory has found application to adsorption (incorporation, partitioning) of charged peptides, hormones, or drugs at the membrane-water interface. Empirically it has been found that an effective charge, smaller than the physical charge, must often be used in the Gouy-Chapman formula. In addition, the large size of these molecules can be expected to influence their adsorption isotherms. To improve evaluation techniques for such experiments, comparatively simple extensions of the standard Gouy-Chapman formalism have been studied which are based on a discrete charge virial expansion. The model allows for the mobility of charged groups at the interface. It accounts for finite size of the adsorbed macromolecules and for discrete charge effects arising from pair interactions in the interface plane. In contrast to previous discrete charge treatments this model nearly coincides with the Gouy-Chapman formalism in the case where the adsorbing molecules are univalent. Large discrepancies are found for multivalent molecules. This could explain the reduced effective charges needed in the standard Gouy-Chapman treatment. The reduction factor can be predicted. The model is mainly limited to low surface coverage, typical for the adsorption studies in question.

Melittin induced voltage-dependent conductance in DOPC lipid bilayers.

Melittin-induced conductance was measured on planar bilayers made from dioleoylphosphatidylcholine. Upon application of a fixed voltage, the current response was monophasic and remained so even after prolonged observation times. The conductance of melittin-doped bilayers increased exponentially with voltage. In addition, an ohmic contribution appeared after some current had passed. The voltage-dependent conductance increased e-fold every 22 mV and was proportional to the fourth power of the aqueous monomeric peptide concentration, for all salt concentrations investigated (0.4-1.8 M NaCl). Discrete conductance steps could be resolved at all these salt concentrations. The amplitudes of these steps were highly variable. In each experiment, conductance was initially only observed for potentials which were positive on the side of peptide addition. As more and more current passed across the bilayer, the current-voltage curves became symmetric. The system needed some time to reach stationary current-voltage characteristics: about 50 min at pH 7 but only about 15 min at pH 8, suggesting involvement of the N-terminus (pK around 7.5) of melittin in the slow formation of a 'prepore'.

Electrostatics of a peptide at a membrane/water interface. The pH dependence of melittin association with lipid vesicles.

The association of the peptide melittin with small unilamellar DMPC vesicles was studied as a function of pH. The results are discussed quantitatively assuming a water-membrane partition equilibrium. Electrostatic surface charging is taken into account as more and more of the strongly basic peptide accumulates at the bilayer/water interface. The data could be well described in terms of a Gouy-Chapman approach involving an effective interfacial charge well below the actual physical charge carried by the individual peptide molecules. The partition coefficient turned out to be pH invariant, so that one can exclude deprotonation reactions upon insertion of the peptide into the bilayer. The effective interfacial charge per associated melittin molecule decreased over a broad range of pH (pH 7 to pH above 10). Contributions of the free amino terminus and of the arginine residues could be determined by comparing with results obtained using modified melittin (N-terminally formylated and fully acetylated). The data suggest approximately equal fractional contributions of the amino terminus and the three lysines to the effective interfacial charge. The two arginines contribute less. Thus, they may be located farther away from the interface or be closely associated with counter-ions. The analysis is extended to the effect of different ionic strengths.

Lipid dependence of peptide-membrane interactions. Bilayer affinity and aggregation of the peptide alamethicin.

Membrane incorporation and aggregation of the peptide alamethicin have been investigated as a function of lipid type. Head group and acyl chain regions both contribute to modulate alamethicin incorporation. Specifically, the peptide prefers thin membranes and saturated chains; incorporation is reduced by the presence of cholesterol. Aggregation of the peptide in the bilayer is virtually insensitive to changes in lipid composition. These findings show some analogies to results obtained with intrinsic membrane proteins and cast doubt on the use of global membrane parameters for interpreting lipid-peptide interactions.

Voltage-dependent pore activity of the peptide alamethicin correlated with incorporation in the membrane: salt and cholesterol effects.

Strong aggregation of incorporated alamethicin in the bilayer of lipid vesicles has been observed spectroscopically at aqueous peptide concentrations above a critical value c*. On the other hand, in conventional gating studies with planar lipid films, the onset of conducting pore formation can be characterized by a threshold voltage V.. We present experimental evidence of a direct correspondence between the effects on c* and V. when these parameters are modulated by adding NaCl (to the aqueous medium) or cholesterol (to the lipid moiety). A quantitative analysis supports the idea that the measured aggregation actually results in pore formation, the voltage-dependence being due to an electric field effect on the partition equilibrium of the peptide between the aqueous and the lipid phases.

Incorporation kinetics in a membrane, studied with the pore-forming peptide alamethicin.

The reaction of fluorescence-labeled alamethicin with unilamellar phospholipid vesicles (DOPC and DMPC) has been investigated in a stopped-flow apparatus. Clearly single exponential time functions have been observed at temperatures above the phase transition of the bilayer. This can be interpreted in terms of an essentially one-step incorporation process. The pseudo first-order forward rate is found to be quite fast, falling in a range somewhat below the diffusion controlled upper bound. The data are quantitatively very well described on the basis of a simple mechanism. This comprises diffusion of peptide into the bilayer accompanied by a more or less slower change of the secondary structure. Aggregation of the incorporated molecules at higher concentrations is indicated to be comparatively rapid.

Alamethicin incorporation in lipid bilayers: a thermodynamic study.

Interaction of the peptide antibiotic alamethicin with phospholipid vesicles has been monitored by changes in its circular dichroic and fluorescent properties. The data are consistent with an incorporation of the peptide in the lipid bilayer. Aggregation of alamethicin in the membrane phase is evident from a characteristic concentration dependence of the incorporation, reflecting the existence of a critical concentration. The data can be fully understood in terms of a theoretical approach that includes aggregation and thermodynamic nonideality. Thermodynamic parameters of the peptide-lipid interaction have been evaluated under a variety of conditions of temperature, ionic strength, and lipid type (saturated and unsaturated fatty acid chains). From the results obtained in this study, one can extrapolate to the incorporation behavior of alamethicin at low concentrations, as they are typical for measurements of conductance across planar lipid films. This leads to a simple explanation of the voltage-gating mechanism of alamethicin in a straightforward way.

Thermodynamic analysis of incorporation and aggregation in a membrane: application to the pore-forming peptide alamethicin.

Interaction of the pore-forming antibiotic alamethicin with small unilamellar vesicles of dioleoylphosphatidylcholine has been studied by means of circular dichroism. The data strongly suggest that alamethicin does not bind to the surface of the vesicles but incorporates into the lipid phase to a fairly large extent. Furthermore, aggregation of the peptide in the membrane is apparent from the existence of a 'critical concentration'. Quantitative evaluation and interpretation of the data rest on a quite generally applicable thermodynamic analysis. The underlying phenomenon is treated in terms of a partition equilibrium between the aqueous and lipid media. In the bilayer phase non-ideal interactions (described by appropriate activity coefficients) as well as aggregate formation are considered. Using this approach the relevant parameters of the alamethicin-lipid system have been determined (yielding, in particular, a partition coefficient of 1.3 X 10(3) for the monomeric peptide and a critical aqueous concentration of 2.5 microM). Finally, the possible relevance of these results for the voltage-dependent gating of alamethicin is briefly pointed out.

Large-ligand adsorption to membranes. III. Cooperativity and general ligand shapes.

In previous reports (Stankowski, S. (1983) Biochim. Biophys. Acta 735, 341-351 and 352-360) the ordinary Scatchard-type analysis has been shown to yield erroneous results when applied to the binding of large molecules to membranes or cells. Formulae have been given to treat the limiting cases of very thin and of very bulky ligands. These results are now extended to include ligands of any shape and cooperative interactions. As an example, data on the cooperative binding of polymyxin to charged lipid bilayers are reevaluated. Adsorption with concomitant incorporation of the large molecule into the membrane is also considered.

Interaction of the HB protein of Bacillus globigii with nucleic acids. Analysis of the binding to DNA and polynucleotides.

Fluorescence of the phenylalanine residues of the HB protein of Baccillus globigii was found to be quenched upon binding to nucleic acids. Using this phenomenon, the binding properties were investigated on the basis of an approach published recently. The stoichiometric numbers were about 10 basepairs per bound protein molecule for double-stranded DNA and about 10 bases for single-stranded polynucleotides, independent of salt concentration. Cooperativity parameters were in the range of 50-250. Binding constants were about 5 X 10(6) M-1 (at 0.1 M NaCl) and decreased with increasing salt concentration. From the salt dependence it is inferred that about one Na+ ion is displaced upon binding of a protein molecule to DNA. Therefore the binding site should contain one positively charged amino acid residue. The protein was found to bind with comparable strength to double-stranded and single-stranded DNA as well as to poly(rA). Hence it is concluded that the HB protein does not belong to the category of 'melting proteins'.

Evaluation of cooperativity for phase transitions in two- and three-dimensional systems.

Use of the so-called "cooperative unit" (readily obtainable from the midpoint slope of phase transition curves) is discussed for the determination of cluster sizes and cooperative interaction energies. This quantity has been commonly employed in a rather empirical way since its correct interpretation is known only for some special cases (linear systems, all-or-none transitions). It is shown in the framework of a lattice model (Ising model) that the cooperative unit may be interpreted in terms of correlation functions and that it defines an average cluster corresponding to the patch size as obtained from scattering experiments. Relations between the cooperative unit and a cooperativity parameter are given for various lattices. Different types of transition curves are discussed using a simple analytical formalism, the quasichemical approximation. Some important nonideality effects are investigated which may lead to a "smearing-out" of first-order transitions.

Linear cooperative binding of large ligands involving mutual exclusion of different binding modes.

A rigorous treatment is given for mutually exclusive multiple mode cooperative binding on a linear structure of equivalent binding "contacts". This will be of special interest with regard to larger ligands implicating the possibility that there are different kinds of binding interactions with more than one monomeric sub unit of a linear biopolymer. Quantitative evaluation of binding properties is shown to be essentially based on calculating the largest root of an algebraic equation. The whole procedure can be practically executed by means of a fairly simple computer program. Various typical examples comprising only two modes are discussed in more detail. For some nucleotide-polylysine systems definite binding parameters have been determined from pertinent experimental data.

Multisite proteins and randomly coiled polymeric ligands. Chain length dependence of binding constants.

A model mechanism was developed for the binding of a rigid multisite protein with a randomly coiled multivalent ligand. Probabilities of the formation of chain loops between sites located at given distances at the protein were calculated by an extension of the concept of ring closure in coiled chain molecules. Expressions were derived for the dependence of overall equilibrium quantities, such as the binding constant between the protein and the ligand, on intrinsic parameters such as intrinsic binding constants, number of sites at the protein and their distances and on the chain length of the polymeric ligand. A pronounced chain length dependence of the overall binding constant was predicted even at chain lengths much longer than the size of the protein. Such a dependence was previously observed for the enzyme prolyl hydroxylase which acts on polymeric substrates like (ProProGly)n. This so far unexplained feature is quantitatively described by the model mechanism which is believed to be applicable to many other interactions of biological importance.