Biomedical subjects

R S Hodges

Publications and source records attributed to R S Hodges.

At least 19 recordsLinked to original sources

PubMed2006

Context-dependent effects on the hydrophilicity/hydrophobicity of side-chains during reversed-phase high-performance liquid chromatography: Implications for prediction of peptide retention behaviour.

The present study set out to investigate whether observed relative hydrophilicity/hydrophobicity values of positively charged side-chains (with Lys and Arg as representative side-chains) or hydrophobic side-chains (with Ile as the representative side-chain) were context-dependent, i.e., did such measured values vary depending on characteristics of the peptides within which such side-chains are substituted (overall peptide hydrophobicity, number of positive charges) and/or properties of the mobile phase (anionic counterions of varying hydrophobicity and concentration)? Reversed-phase high-performance liquid chromatography (RP-HPLC) was applied to two series of four synthetic peptide analogues (+1, +2, +3 and +4 net charge), the only difference between the two peptide series being the substitution of one hydrophobic Ile residue for a Gly residue, in the presence of anionic ion-pairing reagents of varying hydrophobicity (HCOOH approximately H3PO4 < TFA < PFPA < HFBA) and concentration (2-50 mM). RP-HPLC of these peptide series revealed that the relative hydrophilicity of Lys and Arg side-chains in the peptides increased with peptide hydrophobicity. In addition the relative hydrophobicity of Ile decreased dramatically with an increase in the number of positive charges in the peptide, this hydrophobicity decrease being of greater magnitude as the hydrophobicity of the anionic ion-pairing reagent increased. These results have significant implications in the prediction of peptide retention times for proteomic applications.

Amino Acid Sequence↗

PubMed2006

Lateral mobility of proteins in liquid membranes revisited.

The biological function of transmembrane proteins is closely related to their insertion, which has most often been studied through their lateral mobility. For >30 years, it has been thought that hardly any information on the size of the diffusing object can be extracted from such experiments. Indeed, the hydrodynamic model developed by Saffman and Delbrück predicts a weak, logarithmic dependence of the diffusion coefficient D with the radius R of the protein. Despite widespread use, its validity has never been thoroughly investigated. To check this model, we measured the diffusion coefficients of various peptides and transmembrane proteins, incorporated into giant unilamellar vesicles of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) or in model bilayers of tunable thickness. We show in this work that, for several integral proteins spanning a large range of sizes, the diffusion coefficient is strongly linked to the protein dimensions. A heuristic model results in a Stokes-like expression for D, (D proportional, variant 1/R), which fits literature data as well as ours. Diffusion measurement is then a fast and fruitful method; it allows determining the oligomerization degree of proteins or studying lipid-protein and protein-protein interactions within bilayers.

Bacterial Outer Membrane Proteins↗

PubMed2005

Effect of anionic ion-pairing reagent concentration (1-60 mM) on reversed-phase liquid chromatography elution behaviour of peptides.

The homologous series of volatile perfluorinated acids-trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--continue to be excellent anionic ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) after more than two decades since their introduction to this field. It was felt that a thorough, step-by-step re-examination of the effects of anionic ion-pairing reagents over a wide concentration range on RP-HPLC peptide elution behaviour is now due, particularly considering the continuing dominance of such reagents for peptide applications. Thus, RP-HPLC was applied over a range of 1-60 mM phosphoric acid, TFA, PFPA and HFBA to two mixtures of 18-residue synthetic peptides containing either the same net positive charge (+4) or varying positive charge (+1, +2, +3, +4). Peptides with the same charge are resolved very similarly independent of the ion-pairing reagent used, although the overall retention times of the peptides increase with increasing hydrophobicity of the anion: phosphate < TFA- < PFPA- < HFBA-. Peptides of differing charge move at differing rates relative to each other depending on concentration of ion-pairing reagents. All four ion-pairing reagents increased peptide retention time with increasing concentration, albeit to different extents, again based on hydrophobicity of the anion, i.e., the more hydrophobic the anion, the greater the increase in peptide retention time at the same reagent concentration. Interestingly, phosphoric acid produced the best separation of the four-peptide mixture (+1 to +4 net charge). In addition, concentrations above 10 mM HFBA produced a reversal of the elution order of the four peptides (+1 < + 2 < + 3 < + 4) compared to the elution order produced by the other three reagents over the entire concentration range (+4 < + 3 < + 2 < + 1).

Amino Acid Sequence↗

PubMed2005

The perchlorate anion is more effective than the trifluoroacetate anion as an ion-pairing reagent for reversed-phase chromatography of peptides.

The addition of salts, specifically sodium perchlorate (NaClO4), to mobile phases at acidic pH as ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) has been generally overlooked. To demonstrate the potential of NaClO4 as an effective anionic ion-pairing reagent, we applied RP-HPLC in the presence of 0-100 mM sodium chloride (NaCl), sodium trifluoroacetate (NaTFA) or NaClO4 to two mixtures of synthetic 18-residue peptides: a mixture of peptides with the same net positive charge (+4) and a mixture of four peptides of +1, +2, +3 and +4 net charge. Interestingly, the effect of increasing NaClO4 concentration on increasing peptide retention times and selectivity changes was more dramatic than that of either NaCl or NaTFA, with the order of increasing anion effectiveness being Cl- << TFA- < C104-. Such effects were more marked when salt addition was applied to eluents containing 10 mM phosphoric acid (H3PO4) compared to 10 mM trifluoroacetic acid (TFA) due to the lesser starting anion hydrophobicity of the former mobile phase (containing the phosphate ion) compared to the latter (containing the TFA- ion).

Anions↗

PubMed2005

Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography.

Despite the continuing dominance of trifluoroacetic acid (TFA) as the anionic ion-pairing reagent of choice for peptide separations by reversed-phase high-performance liquid chromatography (RP-HPLC), we believe that a step-by-step approach to re-examining the relative efficacy of TFA compared to other ion-pairing reagents is worthwhile, particularly for the design of separation protocols for complex peptide mixtures, e.g., in proteomics applications. Thus, we applied RP-HPLC in the presence of different concentrations of anionic ion-pairing reagents - phosphoric acid, TFA, pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--to a mixture of three groups of four 10-residue peptides, these groups containing peptides of +1, +3 or +5 net charge. Overall separation of the 12-peptide mixture improved with increasing reagent hydrophobicity (phosphate- < TFA- < PFPA- < HFBA-) and/or concentration of the anion, with reagent hydrophobicity having a considerably more pronounced effect than reagent concentration. HFBA, in particular, achieved an excellent separation at a concentration of just 10 mM, whereby the peptides were separated by charged groups (+1 < +3 < +5) and hydrophobicity within these groups. There was an essentially equal effect of reagent hydrophobicity and concentration on each positive charge of the peptides, a useful observation for prediction of the effect of varying counterion concentration hydrophobicity and/or concentration during optimization of peptide purification protocols. Peak widths were greater for the more highly charged peptides, although these could be decreased significantly by raising the acid concentration; concomitantly, peptide resolution increased with increasing concentration of ion-pairing reagent.

Chromatography, High Pressure Liquid↗

PubMed2005

Monomeric and dimeric states exhibited by the kinesin-related motor protein KIF1A.

KIF1A, a kinesin-related motor protein that transports pre-synaptic vesicles in neurons, was originally presumed to translocate along microtubules (MT) as a monomer. Protein structure predictions from its amino acid sequence failed to identify the long coiled-coil domains typical of kinesins, which led researchers to believe it does not oligomerize into the canonical kinesin dimer. However, mounting evidence using recombinant chimeric protein indicates that KIF1A, like conventional kinesin, requires dimerization for fast, unidirectional processive movement along MTs. Because these studies are somewhat indirect, we wished to test the oligomerization state of native KIF1A, and to compare that to full-length recombinant protein. We have performed hydrodynamic analyses to determine the molecular weights of the respective complexes. Our results indicate that most native KIF1A is soluble and indeed monomeric, but recombinant KIF1A is a dimer. MT-binding studies also showed that native KIF1A did not bind to MTs in either the presence of AMP-PNP, apyrase, or adenosine triphosphate (ATP), but recombinant KIF1A bound to MTs most stably in the presence of ATP, indicating very different motor functional states. To further characterize KIF1A's dimerization potential, we prepared peptides corresponding to the neck domains of MmKIF1A and CeUnc104, and by circular dichroism spectroscopy compared these peptides for their ability to form coiled-coils. Interestingly, both MmKIF1A and CeUnc104 neck peptides formed homodimeric coiled-coils, with the MmKIF1A neck coiled-coil exhibiting the greater stability. Collectively, from our data and from previous studies, we predict that native KIF1A can exist as both an inactive monomer and an active homodimer formed in part through its neck coiled-coil domain.

Animals↗

PubMed2004

Monitoring the hydrophilicity/hydrophobicity of amino acid side-chains in the non-polar and polar faces of amphipathic alpha-helices by reversed-phase and hydrophilic interaction/cation-exchange chromatography.

The ability to monitor precisely the hydrophobicity/hydrophilicity effects of amino acid substitutions in both the non-polar and polar faces of amphipathic alpha-helical peptides is critical in such areas as the rational de novo design of more effective antimicrobial peptides. The present study reports our initial results of employing the complementary separation modes of reversed-phase high-performance liquid chromatography (RP-HPLC) and hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) to monitor the effect on apparent peptide hydrophilicity/hydrophobicity and amphipathicity of substituting single L- or D-amino acids into the centre of the non-polar or polar faces of a 26-residue biologically active amphipathic alpha-helical peptide, V681. Our results clearly show that RP-HPLC and HILIC/CEX are best suited for resolving amphipathic peptides where substitutions are made in the non-polar and polar faces, respectively. Further, RP-HPLC and HILIC/CEX were demonstrated to be excellent monitors of hydrophilicity/hydrophobicity variations where amino acid substitutions were made in these respective faces. We believe these complementary high-performance modes offer excellent potential for rational design of novel amphipathic alpha-helical biologically active peptides.

Amino Acids↗

PubMed2004

Optimum concentration of trifluoroacetic acid for reversed-phase liquid chromatography of peptides revisited.

Trifluoroacetic acid (TFA) remains the dominant mobile phase additive for reversed-phase high-performance liquid chromatography (RP-HPLC) of peptides after more than two decades since its introduction to this field. Generally, TFA has been employed in a concentration range of 0.05-0.1% (6.5-13 mM) for the majority of peptide separations. In order to revisit the question as to whether such a concentration range is optimum for separations of peptide mixtures containing peptides of varying net positive charge, the present study examined the effect of varying TFA concentration on RP-HPLC at 25 and 70 degrees C of three groups of synthetic 10-residue synthetic peptides containing either one (+1) or multiple (+3, +5) positively charged groups. The results show that the traditional range of TFA concentrations employed for peptide studies is not optimum for many, perhaps the majority, of peptide applications. For efficient resolution of peptide mixtures, particularly those containing peptides with multiple positive charges, our results show that 0.2-0.25% TFA in the mobile phase will achieve optimum resolution. In addition, the use of high temperature as a complement to such TFA concentration levels is also effective in maximizing peptide resolution.

Amino Acid Sequence↗

PubMed2004

Selectivity differences in the separation of amphipathic alpha-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0: effects of different packings, mobile phase conditions and temperature.

In an ongoing effort to understand the effect of varying reversed-phase high-performance liquid chromatography (RP-HPLC) parameters on the retention behaviour of peptides, necessary for the rational development of separation/optimization protocols, we believe it is important to delineate the contribution of alpha-helical structure to the selectivity of peptide separations. The present study reports the effects of varying column packing, mobile phase conditions and temperature on RP-HPLC retention behaviour at pHs 2.0 and 7.0 of peptides based on the amphipathic peptide sequence Ac-EAEKAAKEXEKAAKEAEK-amide (with position X in the centre of the hydrophobic face of the alpha-helix), where position X is substituted by L- or D-amino acids. At pH 2.0, an increase in trifluoroacetic acid concentration or the addition of sodium perchlorate to a phosphoric acid-based mobile phase had the similar effect of improving peak shape as well as increasing peptide retention time due to ion-pairing effects with the positively-charged peptides; in contrast, at pH 7.0, the addition of salt had little effect save an improvement in peak shape. Temperature was shown to have a complex influence on peptide selectivity due to varying effects on peptide conformation. In addition, subtle effects on peptide selectivity were also noted based on the column packings employed at pHs 2.0 and 7.0.

Amino Acid Sequence↗

PubMed2004

Effects of single D-amino acid substitutions on disruption of beta-sheet structure and hydrophobicity in cyclic 14-residue antimicrobial peptide analogs related to gramicidin S.

Gramicidin S (GS) is a 10-residue cyclic beta-sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure-activity studies of de novo-designed 14-residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single L-amino acid with its corresponding D-enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274, 13181]. The diastereomer with a D-Lys substituted at position 4 caused the greatest improvement in specificity vs. other L to D substitutions within the cyclic 14-residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted beta-sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275, 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a D- or L-amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the D- and L-substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic D-amino acids D-Lys and D-Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the L-diastereomers, which demonstrated strong hemolysis. All of the L-substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the D-substituted peptides tended to improve based on the hydrophilicity of the residue. D-Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic D-amino acid substitutions had superior antimicrobial activity vs. the L-enantiomers although substitution of a hydrophobic D-amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted beta-sheet structure on antimicrobial activity.

1-Naphthylamine↗

PubMed2002

Conformation and interaction of the cyclic cationic antimicrobial peptides in lipid bilayers.

To investigate the role of peptide-membrane interactions in the biological activity of cyclic cationic peptides, the conformations and interactions of four membrane-active antimicrobial peptides [based on Gramicidin S (GS)] were examined in neutral and negatively charged micelles and phospholipid vesicles, using CD and fluorescence spectroscopy and ultracentrifugation techniques. Moreover, the effects of these peptides on the release of entrapped fluorescent dye from unilamellar vesicles of phosphatidylcholine (PC) and phosphatidylethanolamine/phosphatidylglycerol (PE/PG) were studied. The cyclic peptides include GS10 [Cyclo(VKLdYP)2], GS12 [Cyclo(VKLKdYPKVKLdYP)], GS14 [Cyclo(VKLKVdYPLKVKLdYP)] and [d-Lys]4GS14 [Cyclo(VKLdKVdYPLKVKLdYP)] (underlined residues are d-amino acids), were different in their ring size, structure and amphipathicity, and covered a broad spectrum of hemolytic and antimicrobial activities. Interaction of the peptides with the zwitterionic PC and negatively charged PE/PG vesicles were distinct from each other. The hydrophobic interaction seems to be the dominant factor in the hemolytic activity of the peptides, as well as their interaction with the PC vesicles. A combination of electrostatic and hydrophobic interactions of the peptides induces aggregation and fusion in PE/PG vesicles with different propensities in the order: [d-Lys]4GS14 > GS14 > GS12 > GS10. GS10 and GS14 are apparently located in the deeper levels of the membrane interfaces and closer to the hydrophobic core of the bilayers, whereas GS12 and [d-Lys]4GS14 reside closer to the outer boundary of the interface. Because of differing modes of interaction of the cyclic cationic peptides with lipid bilayers, the mechanism of their biological activity (and its relation to peptide-lipid interaction) proved to be versatile and complex, and dependent on the biophysical properties of both the peptides and membranes.

Antimicrobial Cationic Peptides↗

PubMed2002

A possible mechanism for partitioning between homo- and heterodimerization of the yeast homeodomain proteins MATa1 and MATalpha2.

The yeast Saccharomyces cerevisiae has three cell types distinguished by the proteins encoded in their mating-type (MAT) loci: the a and alpha haploids, which express the DNA-binding proteins a1, and alpha1 and alpha2, respectively, and the a/alpha diploid which expresses both a1 and alpha2 proteins. In a/alpha cells, a1-alpha2 heterodimers repress haploid-specific genes and MATalpha1, whereas alpha2 homodimers repress a-specific genes, indicating dual regulatory functions for alpha2 in mating-type control. We previously demonstrated that the two leucine zipper-like coiled-coil motifs, called alpha2A and alpha2B, in the alpha2 N-terminal domain are important to a1-alpha2 heterodimerization. A unique feature of alpha2B is the occurrence of three atypical amino acid residues at a positions within the hydrophobic core. We have conducted mutational analyses of alpha2B peptides and the full-length protein. Our data suggest that these residues may play a critical role in partitioning of the alpha2 protein between heterodimerization with a1 and homodimerization with itself.

Amino Acid Sequence↗

PubMed2002

Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix.

We describe here a systematic study to determine the effect on secondary structure of d-amino acid substitutions in the nonpolar face of an amphipathic alpha-helical peptide. The helix-destabilizing ability of 19 d-amino acid residues in an amphipathic alpha-helical model peptide was evaluated by reversed-phase HPLC and CD spectroscopy. l-Amino acid and d-amino acid residues show a wide range of helix-destabilizing effects relative to Gly, as evidenced in melting temperatures (DeltaTm) ranging from -8.5 degrees C to 30.5 degrees C for the l-amino acids and -9.5 degrees C to 9.0 degrees C for the d-amino acids. Helix stereochemistry stability coefficients defined as the difference in Tm values for the l- and d-amino acid substitutions [(DeltaTm' = TmL and TmD)] ranging from 1 degrees C to 34.5 degrees C. HPLC retention times [DeltatR(XL-XD)] also had values ranging from -0.52 to 7.31 min at pH 7.0. The helix-destabilizing ability of a specific d-amino acid is highly dependent on its side-chain, with no clear relationship to the helical propensity of its corresponding l-enantiomers. In both CD and reversed-phase HPLC studies, d-amino acids with beta-branched side-chains destabilize alpha-helical structure to the greatest extent. A series of helix stability coefficients was subsequently determined, which should prove valuable both for protein structure-activity studies and de novo design of novel biologically active peptides.

Amino Acid Sequence↗

PubMed2001

A polyalanine-based peptide cannot form a stable transmembrane alpha-helix in fully hydrated phospholipid bilayers.

The conformation and amide proton exchangeability of the peptide acetyl-K(2)-A(24)-K(2)-amide (A(24)) and its interaction with phosphatidylcholine bilayers were examined by a variety of physical techniques. When dissolved in or cast from methanol as a dried film, A(24) is predominantly alpha-helical. In aqueous media, however, A(24) exists primarily as a mixture of helical (though not necessarily alpha-helical) and random coiled structures, both of which allow rapid H-D exchange of all amide protons. When incorporated into phospholipids in the absence of water, A(24) also exists primarily as a transmembrane alpha-helix. However, upon hydration of that system, rapid exchange of all amide protons also occurs along with a marked change in the amide I absorption band of the peptide. Also, when dispersed with phosphatidylcholine in aqueous media, the conformation and thermal stability of A(24) are not significantly altered by the presence of the phospholipid or by its gel/liquid-crystalline phase transition. Differential scanning calorimetric and electron spin resonance spectroscopic studies indicate that A(24) has relatively minor effects on the thermodynamic properties of the lipid hydrocarbon chain-melting phase transition, that it does not abolish the lipid pretransition, and that its presence has no significant effect on the orientational order or rates of motion of the phospholipid hydrocarbon chains. We therefore conclude that A(24) has sufficient alpha-helical propensity, but insufficient hydrophobicity, to maintain a stable transmembrane association with phospholipid bilayers in the presence of water. Instead, it exists primarily as a dynamic mixture of helices and other conformers and resides mostly in the aqueous phase where it interacts weakly with the bilayer surface or with the polar/apolar interfacial region of phosphatidylcholine bilayers. Thus, polyalanine-based peptides are not good models for the transmembrane alpha-helical segments of natural membrane proteins.

Calorimetry, Differential Scanning↗

PubMed2001

Mapping the interacting regions between troponins T and C. Binding of TnT and TnI peptides to TnC and NMR mapping of the TnT-binding site on TnC.

Muscular contraction is triggered by an increase in calcium concentration, which is transmitted to the contractile proteins by the troponin complex. The interactions among the components of the troponin complex (troponins T, C, and I) are essential to understanding the regulation of muscle contraction. While the structure of TnC is well known, and a model for the binary TnC.TnI complex has been recently published (Tung, C.-S., Wall, M. E., Gallagher, S. C., and Trewhella, J. (2000) Protein Sci. 9, 1312-1326), very little is known about TnT. Using non-denaturing gels and NMR spectroscopy, we have analyzed the interactions between TnC and five peptides from TnT as well as how three TnI peptides affect these interactions. Rabbit fast skeletal muscle peptide TnT-(160-193) binds to TnC with a dissociation constant of 30 +/- 6 microm. This binding still occurs in the presence of TnI-(1-40) but is prevented by the presence of TnI-(56-115) or TnI-(96-139), both containing the primary inhibitory region of TnI. TnT-(228-260) also binds TnC. The binding site for TnT-(160-193) is located on the C-terminal domain of TnC and was mapped to the surface of TnC using NMR chemical shift mapping techniques. In the context of the model for the TnC.TnI complex, we discuss the interactions between TnT and the other troponin subunits.

Animals↗

PubMed2001

Are trigger sequences essential in the folding of two-stranded alpha-helical coiled-coils?

The amino acid residues comprising the interface between strands of the coiled-coil motif are usually hydrophobic and make a major contribution to coiled-coil folding and stability. However, in some cases the presence of excellent hydrophobic residues at the coiled-coil interface is insufficient for folding. It has been proposed that a "consensus trigger sequence" exists that is necessary within the coiled-coil domains of various proteins to trigger folding. Therefore, in this study we designed a 31-residue hybrid sequence based on sequences from the two-stranded parallel coiled-coil domains of the yeast transcriptional activator GCN4 and the actin-bundling protein Dictyostelium discoideum cortexillin I. The hybrid and its analogs were studied by CD spectroscopy and analytical ultracentrifugation. The hybrid had stable residues in the core "a" and "d" positions in the 3-4 hydrophobic repeat, denoted (abcdefg)n, but did not have a consensus trigger sequence and did not possess appreciable secondary structure as determined by CD spectroscopy. The substitutions in the parent peptide were introduced at positions other than "a" and "d", altering a variety of interactions including alpha-helical propensity, interchain and intrachain electrostatics, and hydrophobicity. Although the substitutions did not bring the overall sequence in closer agreement to the consensus trigger sequence, they increased coiled-coil folding and stability. Therefore, our results suggest that the combination of stabilizing effects along a protein sequence is a more general indicator of protein folding in coiled-coils than the identification of a specific trigger sequence. We propose that surpassing a critical threshold stability value using any type or combination of stabilizing effects will allow coiled-coils to fold, in the absence of a specific trigger sequence per se.

Amino Acid Sequence↗

PubMed2001

Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes.

We have investigated the effect of the presence of 25 mol percent cholesterol on the interactions of the antimicrobial peptide gramicidin S (GS) with phosphatidylcholine and phosphatidylethanolamine model membrane systems using a variety of methods. Our circular dichroism spectroscopic measurements indicate that the incorporation of cholesterol into egg phosphatidylcholine vesicles has no significant effect on the conformation of the GS molecule but that this peptide resides in a range of intermediate polarity as compared to aqueous solution or an organic solvent. Our Fourier transform infrared spectroscopic measurements confirm these findings and demonstrate that in both cholesterol-containing and cholesterol-free dimyristoylphosphatidylcholine liquid-crystalline bilayers, GS is located in a region of intermediate polarity at the polar--nonpolar interfacial region of the lipid bilayer. However, GS appears to be located in a more polar environment nearer the bilayer surface when cholesterol is present. Our (31)P-nuclear magnetic resonance studies demonstrate that the presence of cholesterol markedly reduces the tendency of GS to induce the formation of inverted nonlamellar phases in model membranes composed of an unsaturated phosphatidylethanolamine. Finally, fluorescence dye leakage experiments indicate that cholesterol inhibits the GS-induced permeabilization of phosphatidylcholine vesicles. Thus in all respects the presence of cholesterol attenuates but does not abolish the interactions of GS with, and the characteristic effects of GS on, phospholipid bilayers. These findings may explain why it is more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes while nevertheless disrupting the integrity of the latter at higher peptide concentrations. This additional example of the lipid specificity of GS may aid in the rational design of GS analogs with increased antibacterial but reduced hemolytic activities.

Anti-Bacterial Agents↗

PubMed2001

A differential scanning calorimetric and 31P NMR spectroscopic study of the effect of transmembrane alpha-helical peptides on the lamellar-reversed hexagonal phase transition of phosphatidylethanolamine model membranes.

We have investigated the effects of the model alpha-helical transmembrane peptide Ac-K(2)L(24)K(2)-amide (L(24)) on the thermotropic phase behavior of aqueous dispersions of 1,2-dielaidoylphosphatidylethanolamine (DEPE) to understand better the interactions between lipid bilayers and the membrane-spanning segments of integral membrane proteins. We studied in particular the effect of L(24) and three derivatives thereof on the liquid-crystalline lamellar (L(alpha))-reversed hexagonal (H(II)) phase transition of DEPE model membranes by differential scanning calorimetry and (31)P nuclear magnetic resonance spectroscopy. We found that the incorporation of L(24) progressively decreases the temperature, enthalpy, and cooperativity of the L(alpha)-H(II) phase transition, as well as induces the formation of an inverted cubic phase, indicating that this transmembrane peptide promotes the formation of inverted nonlamellar phases, despite the fact that the hydrophobic length of this peptide exceeds the hydrophobic thickness of the host lipid bilayer. These characteristic effects are not altered by truncation of the side chains of the terminal lysine residues or by replacing each of the leucine residues at the end of the polyleucine core of L(24) with a tryptophan residue. Thus, the characteristic effects of these transmembrane peptides on DEPE thermotropic phase behavior are independent of their detailed chemical structure. Importantly, significantly shortening the polyleucine core of L(24) results in a smaller decrease in the L(alpha)-H(II) phase transition temperature of the DEPE matrix into which it is incorporated, and reducing the thickness of the host phosphatidylethanolamine bilayer results in a larger reduction in the L(alpha)-H(II) phase transition temperature. These results are not those predicted by hydrophobic mismatch considerations or reported in previous studies of other transmembrane alpha-helical peptides containing a core of an alternating sequence of leucine and alanine residues. We thus conclude that the hydrophobicity and conformational flexibility of transmembrane peptides can affect their propensity to induce the formation of inverted nonlamellar phases by mechanisms not primarily dependent on lipid-peptide hydrophobic mismatch.

Calorimetry, Differential Scanning↗