Fluorescence quenching of beta-carboline alkaloids in micellar media. A study to select the adequate surfactant to use in analytical techniques.

The study of fluorescence quenching of the fluorophores allows the localization of the alkaloids (harmane and harmine) in the micelles (SDS, CTAB, Brij-35) to be established. In aqueous micellar solutions (SDS and Brij-35) at pH 13.0, emission corresponding to the neutral or zwitterionic forms can be observed. In the presence of CTAB (pH = 13.0) it was possible to observe the emission of anionic form. These species are not present in buffered aqueous solutions at these pH values. Bromide ion was added to the different surfactant solutions and the quenching effect was studied according to the Stern-Volmer equation. In the presence of SDS the quenching effect is considerably reduced compared to the aqueous solutions without surfactants, while for Brij-35 micelles were similar to those observed in homogeneous aqueous solution. For CTAB micelles a notable fluorescence quenching was observed for the different pH values studied. The fluorescence quenching studies show that the neutral species are associated inside the micelles, instead of the ionic species (cationic, zwitterionic or anionic) remaining on the surface of the micelles. The anionic surface of SDS micelles prevents the quenching effect by anionic quenchers for both neutral and charged species.     

PFG-NMR investigations of the binding of cationic neuropeptides to anionic and zwitterionic micelles

The mechanism by which peptides bind to micelles is believed to be a two-phase process, involving (i). initial electrostatic interactions between the peptide and micelle surface, followed by (ii). hydrophobic interactions between peptide side chains and the micelle core. To better characterize the electrostatic portion of this process, a series of pulse field gradient nuclear magnetic resonance (PFG-NMR) spectroscopic experiments were conducted on a group of neuropeptides with varying net cationic charges (+1 to +3) and charge location to determine both their diffusion coefficients and partition coefficients when in the presence of detergent micelles. Two types of micelles were chosen for the study, namely anionic sodium dodecylsulfate (SDS) and zwitterionic dodecylphosphocholine (DPC) micelles. Results obtained from this investigation indicate that in the case of the anionic SDS micelles, peptides with a larger net positive charge bind to a greater extent than those with a lesser net positive charge (bradykinin > substance P > neurokinin A > Met-enkephalin). In contrast, when in the presence of zwitterionic DPC micelles, the degree of mixed-charge nature of the peptide affects binding (neurokinin A > substance P > Met-enkephalin > bradykinin). Partition coefficients between the peptides and the micelles follow similar trends for both micelle types. Diffusion coefficients for the peptides in SDS micelles, when ranked from largest to smallest, follow a trend where increasing net positive charge results in the smallest diffusion coefficient: Met-enkephalin > neurokinin A > bradykinin > substance P. Diffusion coefficients when in the presence of DPC micelles, when ranked from largest to smallest, follow a trend where the presence of negatively-charged side chains results in the smallest diffusion coefficient: bradykinin > Met-enkephalin > substance P > neurokinin A.     

Cunning Simplicity of a Stoichiometry Driven Protein Folding Thesis

Abstract

Mastoparan B (MP-B) is an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis. NMR spectroscopy was used to study the membrane associated structures of MP-B in various model membrane systems such as 120 mM DPC micelles, 200 mM SDS micelles, and 3%(w/v) DMPC/DHPC (1:2) bicelles. In all systems, MP-B has an amphiphilic α-helical structure from Lys² to Leu¹⁴. NOESY experiments performed on MP-B in nondeuterated SDS micelles show that protons in the indole ring of Trp⁹ are in close contact with methylene protons of SDS micelles. T₁ relaxation data and NOE data revealed that the bound form of MP-B may be dominant in SDS micelles. The interactions between MP-B and zwitterionic DPC micelles were much weaker than those between MP-B and anionic SDS micelles. By substitution of Trp⁹ with Ala⁹, the pore-forming activity of MP-B was decreased dramatically. All of these results imply that strong electrostatic interactions between the positively charged Lys residues in MP-B and the anionic phospholipid head groups must be the primary factor for MP-B binding to the cell membrane. Then, insertion of the indole ring of Trp⁹ into the membrane, as well as the amphiphilic α-helical structures of MP-B may allow MP-B to span the lipid bilayer through the C-terminal portion. These structural features are crucial for the potent antibiotic activities of MP-B.     

Hydrophobic tail length plays a pivotal role in amyloid beta (25–35) fibril–surfactant interactions

The amyloid β‐peptide fragment comprising residues 25–35 (Aβ_25‐35) is known to be the most toxic fragment of the full length Aβ peptide which undergoes fibrillation very rapidly. In the present work, we have investigated the effects of the micellar environment (cationic, anionic, and nonionic) on preformed Aβ_25‐35 fibrils. The amyloid fibrils have been prepared and characterized by several biophysical and microscopic techniques. Effects of cationic dodecyl trimethyl ammonium bromide (DTAB), cetyl trimethylammonium bromide (CTAB), anionic sodium dodecyl sulfate (SDS), and nonionic polyoxyethyleneoctyl phenyl ether (Triton X‐100 or TX) on fibrils have been studied by Thioflavin T fluorescence, UV–vis spectroscopy based turbidity assay and microscopic analyses. Interestingly, DTAB and SDS micelles were observed to disintegrate prepared fibrils to some extent irrespective of their charges. CTAB micelles were found to break down the fibrillar assembly to a greater extent. On the other hand, the nonionic surfactant TX was found to trigger the fibrillation process. The presence of a longer hydrophobic tail in case of CTAB is assumed to be a reason for its higher fibril disaggregating efficacy, the premise of their formation being largely attributed to hydrophobic interactions. Proteins 2016; 84:1213–1223. © 2016 Wiley Periodicals, Inc.     

Relative free energy of binding between antimicrobial peptides and SDS or DPC micelles

We present relative binding free energy calculations for six antimicrobial peptide-micelle systems, three peptides interacting with two types of micelles. The peptides are the scorpion derived antimicrobial peptide (AMP), IsCT and two of its analogues. The micelles are dodecylphosphatidylcholine (DPC) and sodium dodecylsulphate (SDS) micelles. The interfacial electrostatic properties of DPC and SDS micelles are assumed to be similar to those of zwitterionic mammalian and anionic bacterial membrane interfaces, respectively. We test the hypothesis that the binding strength between peptides and the anionic micelle SDS can provide information on peptide antimicrobial activity, since it is widely accepted that AMPs function by binding to and disrupting the predominantly anionic lipid bilayer of the bacterial cytoplasmic membrane. We also test the hypothesis that the binding strength between peptides and the zwitterionic micelle DPC can provide information on peptide haemolytic activities, since it is accepted that they also bind to and disrupt the zwitterionic membrane of mammalian cells. Equilibrium structures of the peptides, micelles and peptide-micelle complexes are obtained from more than 300 ns of molecular dynamics simulations. A thermodynamic cycle is introduced to compute the binding free energy from electrostatic, non-electrostatic and entropic contributions. We find relative binding free energy strengths between peptides and SDS to correlate with the experimentally measured rankings for peptide antimicrobial activities, and relative free energy binding strengths between peptides and DPC to correlate with the observed rankings for peptide haemolytic toxicities. These findings point to the importance of peptide-membrane binding strength for antimicrobial activity and haemolytic activity.     

Conformation of a synthetic antigenic peptide from HIV-1 p24 protein induced by ionic micelles

We studied the interaction of the peptide AAMQMLKETINEEAAEWDRVHPVHAGPIA from the HIV-1 p24 protein in the presence of SDS (anionic) and CTABr (cationic) micelles at pH 7.0 by circular dichroism, fluorescence, and electron spin resonance (ESR). The micelles induced secondary structure as well as a blue shift in the tryptophan fluorescence emission, indicating an interaction between the peptide and the micelles. However, different contents of secondary structure elements were found when the peptide interacts with SDS or CTABr micelles. Steady-state anisotropy indicates a constraint on the rotational mobility of the tryptophan residue of the peptide upon interaction with micelles. ESR studies pointed to different locations for the peptide in either micelle. Our results suggested that at least part of the peptide might be located at the hydrophobic core of the CTABr micelles, probably at the C-terminal region, while it is more inserted into the SDS micelles.     

Deactivation and conformational changes of cutinase in reverse micelles

Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. Copyright 1998 John Wiley & Sons, Inc.     

Ionization and binding equilibria of papaverine in ionic micelles studied by 1H NMR and optical absorption spectroscopy

The binding of the vasodilator drug papaverine (PAV) to micelles of zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS), cationic cetyltrimethylammonium chloride (CTAC) and anionic sodium dodecylsulfate (SDS) in aqueous solution was studied by 1H NMR and electronic absorption spectroscopy. In the presence of HPS or CTAC, the apparent pK(a) of PAV decreased by about 2 units, while it increased by about 2 units upon binding to SDS. However, the chemical shift patterns of both protonated (PAVH+) and deprotonated (PAV0) forms of PAV are not sensitive to the type of surfactant. The association constants were estimated as 5 +/- 2 M(-1) for PAVH+-CTAC, 8 +/- 3 M(-1) for PAVH+-HPS, (7 +/- 2) x 10(5) M(-1) for PAVH+-SDS, and 1.5 x 10(3) to 3.0 x 10(3) M(-1) for the complexes of PAV0 with all three types of micelles. Using these data, an electrostatic potential difference on the micelle-water interface was calculated as 150 +/- 10 mV for CTAC, 140 +/- 10 mV for HPS and - 140 +/- 10 mV for SDS. The results suggest that PAV aromatic rings are located in the hydrophobic part of the micelle. The electrostatic attraction or repulsion of the protonated quinoline nitrogen and surfactant headgroups changes the affinity of PAV to micelles and, thus, shifts the ionization equilibrium of PAV. The electrostatic potential of HPS micellar surface is determined by the cationic dimethylammonium headgroup fragment, whereas the anionic sulfate fragment attenuates the effective charge of HPS headgroup.     

Effect of micellar charge on the conformation and dynamics of melittin

Electrostatic interactions play a crucial role in modulating and stabilizing molecular interactions in membranes and membrane-mimetic systems such as micelles. We have monitored the change in the conformation and dynamics of the cationic hemolytic peptide melittin bound to micelles of various charge types, utilizing fluorescence and circular dichroism (CD) spectroscopy. The sole tryptophan of melittin displays a red-edge excitation shift (REES) of 3-6 nm when bound to anionic, nonionic, and zwitterionic micelles. This suggests that melittin is localized in a restricted environment, probably in the interfacial region of the micelles, and this region offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state tryptophan in melittin. Further, the rotational mobility of melittin is considerably reduced in these micelles and is found to be dependent on the surface charge of micelles. Interestingly, our results show that melittin does not partition into cetyltrimethylammonium bromide (CTAB) micelles owing to electrostatic repulsion between melittin and CTAB micelles, both of which carry a positive charge. In addition, the fluorescence lifetime of melittin is modulated in micelles of different charge types. The lowest mean fluorescence lifetime is observed in the case of melittin bound to anionic sodium dodecyl sulfate (SDS) micelles. CD spectroscopy shows that micelles induce significant helicity to melittin, with maximum helicity being induced in the case of melittin bound to SDS micelles. Fluorescence quenching measurements using the neutral aqueous quencher acrylamide show differential accessibility of melittin in various types of micelles. Taken together, our results show that micellar surface charge can modulate the conformation and dynamics of melittin. These results could be relevant to understanding the role of the surface charge of membranes in the interaction of membrane-active, amphiphilic peptides with membranes.     

Improved detection of rhamnolipid production using agar plates containing methylene blue and cetyl trimethylammonium bromide

Rhamnolipids, produced predominantly by Pseudomonas aeruginosa, are biosurfactants with important applications. For efficient culture screening according to rhamnolipid productivity, the method using agar plates containing methylene blue (MB) and cetyl trimethylammonium bromide (CTAB) was re-examined. An alternative set-up using a fixed underneath light source and image analysis software improved the detection of the circles formed due to complexation between anionic rhamnolipids and cationic MB/CTAB. The roles and effects of MB and CTAB concentrations and pH on the complexation phenomena are reported.     

DETERMINATION OF METHYLENE BLUE BIOSORPTION BY Rhizopus arrhizus IN THE PRESENCE OF SURFACTANTS WITH DIFFERENT CHEMICAL STRUCTURES

Methylene blue (MB) biosorption properties of Rhizopus arrhizus were investigated in the presence of surfactants. The effects of cationic and anionic surfactants on MB removal by dead biomass (1 g L^?1) were determined. MB removal was tested as a function of initial pH (2–12), contact time (5–1440 min), and dye (37.4–944.7 mg L^?1) and surfactant (0–10 mM) concentrations. The opposite charged anionic surfactant dodecylbenzenesulfonic acid sodium salt (DBS) enhanced sorption of cationic MB by biomass dramatically. Maximum biosorption capacity was 471.5 mg g^?1 at pH 8 with 0.5 mM DBS at 944.7 mg L^?1 MB concentration. The surfactant-stimulated fungal decolorization method may provide a highly efficient, inexpensive, and time-saving procedure in biological wastewater treatment technologies.     

The interaction of beta-amyloid protein fragment (12-28) with lipid environments.

The neurotoxicity of beta-amyloid protein (beta AP) fragments may be a result of their solution conformation, which is very sensitive to solution conditions. In this work we describe NMR and CD studies of the conformation of beta AP(12-28) in lipid (micelle) environments as a function of pH and lipid type. The interaction of beta AP(12-28) with zwitterionic dodecylphosphocholine (DPC) micelles is weak and alters the conformation when compared to water solution alone. By contrast, the interaction of the peptide with anionic sodium dodecylsulfate (SDS) micelles is strong: beta AP(12-28) is mostly bound, is alpha-helical from K16 to V24, and aggregates slowly. The pH-dependent conformation changes of beta AP(12-28) in solution occur in the pH range at which the side-chain groups of E22, D23, H13, and H14 are deprotonated (pKas ca. 4 and 6.5); the interaction of beta AP(12-28) with SDS micelles alters the pH-dependent conformational transitions of the peptide whereas the weak interaction with DPC micelles causes little change.     

Evidence for a folded structure of Met-enkephalin in membrane mimetic systems: 1H-nmr studies in sodiumdodecylsulfate, lyso-phosphatidylcholine, and mixed lyso-phosphatidylcholine/sulfatide micelles

¹H-nmr spectra of Met-enkephalin dissolved in aqueous solution of sodiumdodecylsulfate (SDS) micelles are reported as a function of pH and temperature. The temperature behavior of the amide protons is compared with that observed for the same peptide dissolved in aqueous solution of lyso-phosphatidylcholine (LPC) and lyso-phosphatidylcholine-sulfatide (LPC-SH) micelles. The temperature coefficients are affected by the micelle polarity, which suggests that the peptide backbone is not remote from the micelle surface. pH titration performed in the presence of SDS micelles gives a number of intrinsic and extrinsic pK_a values, indicative of a folded structure of the opioid molecule. This conformation is characterized by the existence of an intramolecular hydrogen bond involving the Met-5 amide proton and an interaction of the N-terminal residue with the aliphatic side chains of both Phe-4 and Met-5.     

Functional polymer brushes via surface-initiated atom transfer radical graft polymerization for combating marine biofouling

Dense and uniform polymer brush coatings were developed to combat marine biofouling. Nonionic hydrophilic, nonionic hydrophobic, cationic, anionic and zwitterionic polymer brush coatings were synthesized via surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-hydroxyethyl methacrylate, 2,3,4,5,6-pentafluorostyrene, 2-(methacryloyloxy)ethyl trimethylammonium chloride, 4-styrenesulfonic acid sodium and N,N′-dimethyl-(methylmethacryloyl ethyl) ammonium propanesulfonate, respectively. The functionalized surfaces had different efficacies in preventing adsorption of bovine serum albumin (BSA), adhesion of the Gram-negative bacterium Pseudomonas sp. NCIMB 2021 and the Gram-positive Staphylococcus aureus, and settlement of cyprids of the barnacle Amphibalanus amphitrite (=Balanus amphitrite). The nonionic hydrophilic, anionic and zwitterionic polymer brushes resisted BSA adsorption during a 2 h exposure period. The nonionic hydrophilic, cationic and zwitterionic brushes exhibited resistance to bacterial fouling (24 h exposure) and cyprid settlement (24 and 48 h incubation). The hydrophobic brushes moderately reduced protein adsorption, and bacteria and cyprid settlement. The anionic brushes were least effective in preventing attachment of bacteria and barnacle cyprids. Thus, the best approach to combat biofouling involves a combination of nonionic hydrophilic and zwitterionic polymer brush coatings on material surfaces.     

Physico-chemical studies of micelle formation on sepia cartilage collagen solutions in acetate buffer and its interaction with ionic and nonionic micelles. Hydrodynamic and thermodynamic studies

Sepia cartilage collagen (pepsin-extracted) in acetate buffer (pH = 2.98) forms micelles at a particular concentration below which they do not normally form. The critical micelle concentration (cmc) of the collagen was determined in buffer as well as in SDS, cetyltrimethylammonium bromide (CTAB) and Tween-80 micellar environments at different temperatures. Mutual interaction of collagen micelles with the ionic and nonionic micelles through the formation of the mixed micelle concept has also been found. The cmc of collagen decreased in the presence of SDS and Tween-80 micelles whereas it increased in the presence of CTAB micelles. This clearly suggests that the micelle formation of collagen is facilitated by the presence of SDS and Tween-80 and hindered by CTAB micelles. The various thermodynamic parameters were estimated from viscosity measurements and the transfer of collagen into the micelles of various surfactants and the reverse phenomenon was analyzed. This analysis has also been modelled conceptually as a different phase and the results have supported the above phenomenon. Our thermodynamic results are also able to predict the exact denaturation temperature as well as the structural order of water in the collagen in various environments. The hydrated volumes, Vh, of collagen in the above environments and intrinsic viscosity were also calculated. The low intrinsic viscosity, [eta], of collagen in an SDS environment compared to buffer and other surfactant environments suggested more workable systems in cosmetic and dermatological skin care preparations. The one and two-hydrogen-bonded models of this collagen in various environments have been analyzed. The calculated thermodynamic parameters varied with the concentration of collagen. The change of thermodynamic parameters from coil-coil to random-coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results. Thermodynamic results suggest that the stability of the collagen in the additive environments is in the following order: SDS greater than Tween-80 greater than buffer greater than CTAB.     

Protein unfolding in detergents: effect of micelle structure,ionic strength,pH, and temperature

The 101-residue monomeric protein S6 unfolds in the anionic detergent sodium dodecyl sulfate (SDS) above the critical micelle concentration, with unfolding rates varying according to two different modes. Our group has proposed that spherical micelles lead to saturation kinetics in unfolding (mode 1), while cylindrical micelles prevalent at higher SDS concentrations induce a power-law dependent increase in the unfolding rate (mode 2). Here I investigate in more detail how micellar properties affect protein unfolding. High NaCl concentrations, which induce cylindrical micelles, favor mode 2. This is consistent with our model, though other effects such as electrostatic screening cannot be discounted. Furthermore, unfolding does not occur in mode 2 in the cationic detergent LTAB, which is unable to form cylindrical micelles. A strong retardation of unfolding occurs at higher LTAB concentrations, possibly due to the formation of dead-end protein-detergent complexes. A similar, albeit much weaker, effect is seen in SDS in the absence of salt. Chymotrypsin inhibitor 2 exhibits the same modes of unfolding in SDS as S6, indicating that this type of protein unfolding is not specific for S6. The unfolding process in mode 1 has an activation barrier similar in magnitude to that in water, while the activation barrier in mode 2 is strongly concentration-dependent. The strong pH-dependence of unfolding in SDS and LTAB suggests that the rate of unfolding in anionic detergent is modulated by repulsion between detergent headgroups and anionic side chains, while cationic side chains modulate unfolding rates in cationic detergents.     

Studies on heme release from normal and metal ion reconstituted hemoglobin mediated through ionic surfactant

The interaction of metal-substituted hemoglobin (MHb), where M = Ni and Cu (T-state with no O2 and CO binding capability) and Fe (R-state when CO is bound), with cationic cityl trimethyl ammonium bromide (CTAB) and anionic (sodium dodecyl sulfate-SDS) surfactants has been studied using spectroscopic techniques-UV-visible, electron paramagnetic resonance (EPR), and Fourier transform-Raman-with additional supportive evidence coming from conductivity measurements. We observed the loss of 5-coordination in all three hemoglobins below the critical micelle concentration (CMC) of surfactant, with noticeable differences, suggesting differing mechanisms involved in this process. In addition, above the CMC, Ni- and Cu-hemes were found to leave their proteins more easily than Fe-heme, presumably due to weaker or no bond with the proximal histidine in the former. The released heme is stabilized by micellar media through a hydrophobic interaction process. Of the two surfactants, CTAB seems to be capable of releasing the heme better than SDS and it is attributed to the greater hydrophobicity of CTAB though the charge of the surfactant plays an important role.     

The minimal structural requirement of concanavalin A that retains its functional aspects

A systematic investigation of the effects of several commonly used detergents on the conformation and function of concanavalin A at pH 7 in solution form was made by using circular dichroism (CD), intrinsic fluorescence, 1-anilino 8-sulphonic acid (ANS) binding, dynamic light scattering (DLS) and sugar inhibition assay. In the presence of 6.0 mM sodium dodecyl sulphate (SDS), an anionic detergent, and 0.8 mM cetyl tri methyl ammonium bromide (CTAB), a cationic detergent, intermediate states of concanavalin A were obtained having a negative CD peaks at 222 and 208 nm respectively, a characteristic of alpha-helix. These states also retained tertiary contacts with altered tryptophan environment and high ANS binding (exposed hydrophobic area) which can be characterized as molten globule states. Concanavalin A in the presence of 5.0 mM 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulphonate (CHAPS), a zwitterionic detergent, and 0.07 mM brij-35, a non-ionic detergent, also exists in intermediate states. These intermediates (molten globules) had high ANS binding with native-like secondary (inherent beta-sheet) and tertiary structure. The intermediate states were characterized further by means of dynamic light-scattering measurements and kinetic data. To study the possible functional requirement of the minimum structure, the intermediate states characterized in the presence of detergents were shown to retain the activity with polysaccharide (dextran). The pattern of activity observed was brij-35 > CHAPS > CTAB > SDS. The specific binding and activity of concanavalin A with ovalbumin was investigated as a function of time by turbidity measurements. Cationic and anionic detergents showed significant effects on the structure of concanavalin A as compared with zwitterionic and non-ionic detergents.     

Influence of Ionic Surfactants on the Microstructure of Heat-Set β-Lactoglobulin-Stabilized Emulsion Gels

Using confocal microscopy, we studied the effect of heating (up to 85°C) on the microstructure of β-lactoglobulin-stabilized emulsions (20 vol% oil, pH 6.8) containing excess protein (total protein content 13.2%). Two different fluorescent dyes were used to separately visualize the oil droplets and the protein. In overlay micrographs, their location with respect to each other could then be determined. In the presence of a low salt concentration, flocculation of the emulsion without surfactant was inhibited, by a mechanism analogous to the “salting-in” of aqueous protein solutions. Addition of the anionic surfactant sodium dodecyl sulfate (SDS) caused weak flocculation, probably as a result of the formation of protein−SDS complexes. The final heat-set emulsion contained distinct pores for a surfactant/protein ratio of R = 1, but no pores for R = 2. Addition of the cationic surfactant cetyl trimethyl ammonium bromide (CTAB) caused strong aggregation, as indicated by microscopic observation of the concentrated emulsion and light scattering of the diluted emulsion. For R = 1 with CTAB, there were aggregates consisting of oil droplets and excess protein. At R = 2, almost all the excess protein was aggregated into separate protein flakes. In the final emulsion gels containing CTAB, the protein was more spread out. Differing structural behavior with anionic and cationic surfactants has been interpreted in terms of different protein−surfactant interactions in aqueous solution and at the oil−water interface, both before and after protein denaturation.     

Antimicrobial peptide RP-1 structure and interactions with anionic versus zwitterionic micelles

Topologically, platelet factor-4 kinocidins consist of distinct N-terminal extended, C-terminal helical, and interposing gamma-core structural domains. The C-terminal alpha-helices autonomously confer direct microbicidal activity, and the synthetic antimicrobial peptide RP-1 is modeled upon these domains. In this study, the structure of RP-1 was assessed using several complementary techniques. The high-resolution structure of RP-1 was determined by NMR in anionic sodium dodecyl sulfate (SDS) and zwitterionic dodecylphosphocholine (DPC) micelles, which approximate prokaryotic and eukaryotic membranes, respectively. NMR data indicate the peptide assumes an amphipathic alpha-helical backbone conformation in both micelle environments. However, small differences were observed in the side-chain orientations of lysine, tyrosine, and phenylalanine residues in SDS versus DPC environments. NMR experiments with a paramagnetic probe indicated differences in positioning of the peptide within the two micelle types. Molecular dynamics (MD) simulations of the peptide in both micelle types were also performed to add insight into the peptide/micelle interactions and to assess the validity of this technique to predict the structure of peptides in complex with micelles. MD independently predicted RP-1 to interact only peripherally with the DPC micelle, leaving its spherical shape intact. In contrast, RP-1 entered deeply into and significantly distorted the SDS micelle. Overall, the experimental and MD results support a preferential specificity of RP-1 for anionic membranes over zwitterionic membranes. This specificity likely derives from differences in RP-1 interaction with distinct lipid systems, including subtle differences in side chain orientations, rather than gross changes in RP-1 structure in the two lipid environments.