Lysophospholipid-Containing Membranes Modulate the Fibril Formation of the Repeat Domain of a Human Functional Amyloid,Pmel17

Pmel17 is an important protein for pigmentation in human skin and eyes. Proteolytic fragments from Pmel17 form fibrils upon which melanin is deposited in melanosomes. The repeat domain (RPT) derived from Pmel17 only forms fibrils under acidic melanosomal conditions. Here, we examined the effects of lipids on RPT aggregation to explore whether intramelanosomal vesicles can facilitate fibrillogenesis. Using transmission electron microscopy, circular dichroism, and fluorescence spectroscopy, we monitored fibril formation at the ultrastructural, secondary conformational, and local levels, respectively. Phospholipid vesicles and lysophospholipid (lysolipid) micelles were employed as membrane mimics. The surfactant-like lysolipids are particularly pertinent due to their high content in melanosomal membranes. Interestingly, RPT aggregation kinetics were influenced only by lysolipid-containing phospholipid vesicles. While both vesicles containing either anionic lysophosphatidylglycerol (LPG) or zwitterionic lysophosphatidylcholine (LPC) stimulate aggregation, LPG exerted a greater effect on reducing the apparent nucleation time. A detailed comparison showed distinct behaviors of LPG versus LPC monomers and micelles plausibly originating from their headgroup hydrogen bonding capabilities. Acceleration and retardation of aggregation were observed for LPG monomers and micelles, respectively. Because a specific interaction between LPG and RPT was identified by intrinsic W423 fluorescence and induced α-helical structure, it is inferred that binding of LPG near the C-terminal amyloid core initiates intermolecular association, whereas stabilization of α-helical conformation inhibits β-sheet formation. Contrastingly, LPC promotes RPT aggregation at both submicellar and micellar concentrations via non-specific binding with undetectable secondary structural change. Our findings suggest that protein–lysolipid interactions within melanosomes may regulate amyloid formation in vivo.     

Induction,inhibition, and incorporation: Different roles for anionic and zwitterionic lysolipids in the fibrillation of the functional amyloid FapC

Amyloid proteins are widespread in nature both as pathological species involved in several diseases and as functional entities that can provide protection and storage for the organism. Lipids have been found in amyloid deposits from various amyloid diseases and have been shown to strongly affect the formation and structure of both pathological and functional amyloid proteins. Here, we investigate how fibrillation of the functional amyloid FapC from Pseudomonas is affected by two lysolipids, the zwitterionic lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine and the anionic lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LPG). Small-angle X-ray scattering, circular dichroism, dynamic light scattering, and thioflavin T fluorescence measurements were performed simultaneously on the same sample to ensure reproducibility and allow a multimethod integrated analysis. We found that LPG strongly induces fibrillation around its critical micelle concentration (cmc) by promoting formation of large structures, which mature via accumulation of intermediate fibril structures with a large cross section. At concentrations above its cmc, LPG strongly inhibits fibrillation by locking FapC in a core–shell complex. In contrast, lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine induces fibrillation at concentrations above its cmc, not via strong interactions with FapC but by being incorporated during fibrillation and likely stabilizing the fibrillation nucleus to reduce the lag phase. Finally, we show that LPG is not incorporated into the fibril during assembly but rather can coat the final fibril. We conclude that lipids affect both the mechanism and outcome of fibrillation of functional amyloid, highlighting a role for lipid concentration and composition in the onset and mechanism of fibrillation in vivo.     

Tau Fibrillation Induced by Heparin or a Lysophospholipid Show Different Initial Oligomer Formation

The protein tau is involved in several neurogenerative diseases such as Alzheimer’s Disease, where tau content and fibrillation have been linked to disease progression. Tau colocalizes with phospholipids and glycosaminoglycans in vivo. We investigated if and how tau fibrillation can be induced by two lysophospholipids, namely the zwitterionic 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC) and the anionic 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LPG) as well as the glycosaminoglycan heparin. We used a range of biophysical techniques including small-angle X-ray scattering, Thioflavin T fluorescence, and SDS-PAGE, collecting data at various time points to obtain structural information on each phase of the fibrillation. We find that LPC does not induce fibrillation or interact with tau. Low concentrations of LPG induce fibrillation by formation of small hydrophobic clusters with monomeric tau. At higher LPG concentrations, a core–shell complex is formed where tau wraps around LPG micelles with regions extending away from the micelles. For heparin, loosely associated oligomers are formed rapidly with around ten tau molecules. Fibrils formed with either LPG or heparin show similar final cross-section dimensions. Furthermore, SDS-resistant oligomers are observed for both LPG and heparin. Our study demonstrates that tau fibrillation can be induced by two different biologically relevant cofactors leading to structurally different initial states but similar cross-sectional dimensions for the fibrils. Structural information about initial states prior to fibril formation is important both to gain a better understanding of the onset of fibrillation in vivo, and for the development of targeted drugs that can reduce or abolish tau fibrillation.     

Folding of outer membrane protein A in the anionic biosurfactant rhamnolipid

Folding and stability of bacterial outer membrane proteins (OMPs) are typically studied in vitro using model systems such as phospholipid vesicles or surfactant. OMP folding requires surfactant concentrations above the critical micelle concentration (cmc) and usually only occurs in neutral or zwitterionic surfactants, but not in anionic or cationic surfactants. Various Gram-negative bacteria produce the anionic biosurfactant rhamnolipid. Here we show that the OMP OmpA can be folded in rhamnolipid at concentrations above the cmc, though the thermal stability is reduced compared to the non-ionic surfactant dodecyl maltoside. We discuss implications for possible interactions between OMPs and biosurfactants in vivo.     

Modulation of cutinase stability and structure by phospholipid detergents

Fusarium solani pisi cutinase hydrolyses triglycerides of different lengths. Here we show that micelle-forming short-chain (C6-C9) phospholipids significantly reduce cutinase stability (both below and above the critical micelle concentration cmc) and rates of folding (only above cmc), trapping cutinase in an inactive state which only regains activity over hours to days, rather than the few seconds required for refolding in the absence of detergent. Destabilization decreases with increasing chain length, and increases with cmc, indicating that monomers and micelles cooperate in destabilizing cutinase. Detergents have little effect on enzymatic activity and confer no changes in secondary structure. Some changes in chemical shift occur around the enzyme active site, although distant regions are also affected. To our knowledge, this is the first example of marked destabilization of a water-soluble protein by zwitterionic detergents, highlighting the multitude of different detergent interactions with enzymes that target amphiphilic substrates and providing means of trapping a protein in a metastable state. We propose a model for destabilization where monomers via various binding sites on the native state prime it for interacting with micelles in a destabilizing fashion, whereas only micelles halt refolding due to the absence of these monomer-binding sites in the denatured state.     

Repeat domains of melanosome matrix protein Pmel17 orthologs form amyloid fibrils at the acidic melanosomal pH

Most amyloids are pathological, but fragments of Pmel17 form a functional amyloid in vertebrate melanosomes essential for melanin synthesis and deposition. We previously reported that only at the mildly acidic pH (4-5.5) typical of melanosomes, the repeat domain (RPT) of human Pmel17 can form amyloid in vitro. Combined with the known presence of RPT in the melanosome filaments and the requirement of this domain for filament formation, we proposed that RPT may be the core of the amyloid formed in vivo. Although most of Pmel17 is highly conserved across a broad range of vertebrates, the RPT domains vary dramatically, with no apparent homology in some cases. Here, we report that the RPT domains of mouse and zebrafish, as well as a small splice variant of human Pmel17, all form amyloid specifically at mildly acid pH (pH ～5.0). Protease digestion, mass per unit length measurements, and solid-state NMR experiments suggest that amyloid of the mouse RPT has an in-register parallel β-sheet architecture with two RPT molecules per layer, similar to amyloid of the Aβ peptide. Although there is no sequence conservation between human and zebrafish RPT, amyloid formation at acid pH is conserved.     

Probing fibril dissolution of the repeat domain of a functional amyloid, Pmel17, on the microscopic and residue level

Pmel17 is a human amyloid involved in melanin synthesis. A fragment of Pmel17, the repeat domain (RPT) rich in glutamic acids, forms amyloid only at mildly acidic pH. Unlike pathological amyloids, these fibrils dissolve at neutral pH, supporting a reversible aggregation-disaggregation process. Here, we study RPT dissolution using atomic force microscopy and solution-state nuclear magnetic resonance spectroscopy. Our results reveal asymmetric fibril disassembly proceeding in the absence of intermediates. We suggest that fibril unfolding involves multiple deprotonation events resulting in electrostatic charge repulsion and filament dissolution.     

Segmental polymorphism in a functional amyloid

Although amyloid fibrils are generally considered to be causative or contributing agents in amyloid diseases, several amyloid fibrils are also believed to have biological functions. Among these are fibrils formed by Pmel17 within melanosomes, which act as a template for melanin deposition. We use solid-state NMR to show that the molecular structures of fibrils formed by the 130-residue pseudo-repeat domain Pmel17:RPT are polymorphic even within the biologically relevant pH range. Thus, biological function in amyloid fibrils does not necessarily imply a unique molecular structure. Solid-state NMR spectra of three Pmel17:RPT polymorphs show that in all cases, only a subset (∼30%) of the full amino acid sequence contributes to the immobilized fibril core. Although the repetitive nature of the sequence and incomplete spectral resolution prevent the determination of unique chemical shift assignments from two- and three-dimensional solid-state NMR spectra, we use a Monte Carlo assignment algorithm to identify protein segments that are present in or absent from the fibril core. The results show that the identity of the core-forming segments varies from one polymorph to another, a phenomenon known as segmental polymorphism.     

Size control of styrene oxide-ethylene oxide diblock copolymer aggregates with classical surfactants: DLS, TEM, and ITC study

The interactions between the diblock copolymer S(15)E(63) and the surfactants sodium dodecyl sulfate (SDS), sodium decyl sulfate (SDeS), and sodium octyl sulfate (SOS) have been investigated by dynamic light scattering (DLS), transmission electron microscopy (TEM), and isothermal titration calorimetry (ITC). The surfactants with the same headgroup differentiate in their chain length. At 20 degrees C, the block copolymer is associated into micelles with a hydrodynamic radius of 11.6 nm, which is composed of a hydrophobic styrene oxide (S) core and a water-swollen oxypolyethylene (PEO or E) corona. The different copolymer/surfactant systems have been studied at a constant copolymer concentration of 2.5 g dm(-3) and in a vast range of surfactant concentrations, from 7.5 x 10(-6) up to 0.75 M. When SDS and SDeS are added to the block copolymer solution, different regions are observed in the DLS data: at low surfactant concentrations (c < 1.0 x 10(-4) M), single surfactant molecules associate with the copolymer micelle, probably the former being solubilized in the micelle core, leading to a certain disruption of the mixed micelle due to repulsive electrostatic interactions between surfactant headgroups followed by a stabilization of the mixed micelle. At higher concentrations (1.0 x 10(-4) < c < 0.1 M), two types of copolymer-surfactant complexes coexist: one large copolymer-rich/surfactant complex and one small complex consisting of one or a few copolymer chains and rich in surfactants. At higher SDS and SDeS concentrations, complete disintegration of mixed micelles takes place. In contrast, SOS-S(15)E(63) interactions are less important up to surfactant concentrations of 0.05 M due to its higher hydrophilicity, reducing the hydrophobic interactions between surfactant alkyl chains and copolymer micelles. At concentration larger than the critical aggregation concentration (cac) of the system, 0.05 M, disruption of copolymer micelles occurs. These regions have been confirmed by transmission electron microscopy. On the other hand, the titration calorimetric data for SDS and SDeS present an endothermic increase indicating the formation of mixed copolymer-rich-surfactant micelles. From that point, important differences in the ITC plot for both surfactants are present. However, the ITC curve obtained after titration of a SOS solution in the copolymer solution is quite similar to that of its titration in water.     

Determining the effect of the incorporation of unnatural amino acids into antimicrobial peptides on the interactions with zwitterionic and anionic membrane model systems

Circular Dichroism (CD), isothermal calorimetry (ITC) and calcein fluorescence leakage experiments were conducted to provide insight into the mechanisms of binding of a series of antimicrobial peptides containing unnatural amino acids (Ac-XF-Tic-Oic-XK-Tic-Oic-XF-Tic-Oic-XK-Tic-KKKK-CONH₂) to zwitterionic and anionic micelles, SUVs and LUVs; where X (Spacer# 1) is either Gly, β-Ala, Gaba or 6-aminohexanoic acid. It is the intent of this investigation to correlate these interactions with the observed potency and selectivity against several different strains of bacteria. The CD spectra of these compounds in the presence of zwitterionic DPC micelles and anionic SDS micelles are very different indicating that these compounds adopt different conformations on binding to the surface of anionic and zwitterionic membrane models. These compounds also exhibited very different CD spectra in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG SUVs and LUVs, indicating the formation of different conformations on interaction with the two membrane types. This observation is also supported by ITC and calcein leakage data. ITC data suggested these peptides interact primarily with the surface of zwitterionic LUVs and was further supported by fluorescence experiments where the interactions do not appear to be concentration dependent. In the presence of anionic membranes, the interactions appear more complex and the calorimetric and fluorescence data both imply pore formation is dependent on peptide concentration. Furthermore, evidence suggests that as the length of Spacer# 1 increases the mechanism of pore formation also changes. Based on the observed differences in the mechanisms of interactions with zwitterionic and anionic LUVs these AMPs are potential candidates for further drug development.     

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC(1)]-Gm; [TOAC(1), Ser(2,6,11,15)]-Gm; [Trp(7)]-Gm; [Ser(2,6,11,15), Trp(7)]-Gm; [Trp(9)]-Gm; and [Ser(2,6,11,15), Trp(9)]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a beta-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC(1)]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.     

Role of Co-Solute in Biomolecular Stability: Glucose, Urea and the Water Structure

We have studied the action of urea and glucose on the stability of DNAand micelles. We measured the melting temperature of aqueous solutionsof DNA with urea or glucose as a co-solute; we have also measured thechanges in the critical micelle concentrations (cmc) of Sodium DodecylSulfate and Triton X-100 by addition of urea and glucose. Ourexperimental results show that glucose increases the melting temperature ofDNA and decreases the cmc, while urea acts in the contrary direction. The effects of urea and glucose on the stability of DNA and micelles canbe explained by the weakening and enhancement of hydrophobicinteractions, respectively. These effects on hydrophobic interactions arediscussed in this paper.     

Why Study Functional Amyloids? Lessons from the Repeat Domain of Pmel17

One of the current challenges facing biomedical researchers is the need to develop new approaches in preventing amyloid formation that is associated with disease. While amyloid is generally considered detrimental to the cell, examples of amyloids that maintain a benign nature and serve a specific function exist. Here, we review our work on the repeat domain (RPT) of the functional amyloid Pmel17. Specifically, the RPT domain contributes in generating amyloid fibrils in melanosomes upon which melanin biosynthesis occurs. Amyloid formation of RPT was shown to be pH sensitive, aggregating only under acidic conditions associated with melanosomal pH. Furthermore, preformed fibrils rapidly dissolved at neutral pH to generate benign monomeric species. From a biological perspective, this unique reversible aggregation/disaggregation is a safeguard against an event of releasing RPT fibrils in the cytosol, resulting in rapid fibril unfolding and circumventing cytotoxicity. Understanding how melanosomes preserve a safe environment will address vital questions that remain unanswered with pathological amyloids.     

Endothelins specifically recognize lysophosphatidylcholine micelles

Lysophosphatidylcholine (LPC), a major phospholipid component of oxidized low‐density lipoprotein (ox‐LDL), is implicated in numerous inflammatory diseases, including atherosclerosis. Here, to clarify the relationship between bioactive endothelins (ETs) (which are considered to be potent proinflammatory mediators) and LPC/ox‐LDL, we investigated the interaction between ETs and LPC/ox‐LDL by fluorescence spectroscopy and western blotting. Tryptophan fluorescence measurements revealed ETs specifically interacted with LPC at concentrations that exceeded the critical micelle concentration (CMC). The tryptophan residue in ETs was not likely to be involved directly in the interaction between ETs and LPC micelles. Tryptophan fluorescence quenching revealed tryptophan residue in ETs where LPC concentrations were below the CMC may be buried deeply in the peptide or may interact with other amino acid residues, whereas tryptophan residue in ETs in the presence of LPC at concentrations exceeding the CMC was exposed outside of the peptide. Furthermore, ETs bind to ox‐LDL in a concentration‐dependent manner. These results strongly suggest that ox‐LDL contains micelle‐rich LPCs and that ETs specifically interact with the bioactive LPC micelles. Further study of the interaction between ETs and LPC micelles contained in ox‐LDL will provide important information on the development and progression of many inflammatory diseases, including atherosclerosis. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Amyloid Fibrillation of Insulin under Water-Limited Conditions

Amyloid fibrillation in water-organic mixtures has been widely studied to understand the effect of protein-solvent interactions on the fibrillation process. In this study, we monitored insulin fibrillation in formamide and its methyl derivatives (formamide, N-methyl formamide, N,N-dimethyl formamide) in the presence and absence of water. These model solvent systems mimic the cellular environment by providing denaturing conditions and a hydrophobic environment with limited water content. Thioflavin T (ThT) assay revealed that binary mixtures of water with formamide and its methyl derivatives enhanced fibrillation rates and β-sheet abundance, whereas organic solvents suppressed insulin fibrillation. We utilized solution small-angle x-ray scattering (SAXS) and differential scanning calorimetry (DSC) to investigate the correlation between protein-solvent interactions and insulin fibrillation. SAXS experiments combined with simulated annealing of the protein indicated that the degree of denaturation of the hydrophobic core region at residues B11–B17 determines the fibrillation rate. In addition, DSC experiments suggested a crucial role of hydrophobic interactions in the fibrillation process. These results imply that an environment with limited water, which imitates a lipid membrane system, accelerates protein denaturation and the formation of intermolecular hydrophobic interactions during amyloid fibrillation.     

Gelatin-alpha olefin sulfonate interactions studied by dynamic light scattering

Dynamic light scattering (DLS) measurements were performed to study the binding of anionic surfactant alpha olefin sulfonate (AOS) to gelatin chains at various NaCl concentrations at 30 degrees C in aqueous sodium phosphate buffer (pH = 6.8) solutions. The surfactant concentration was varied from 0 to 80 mM and the NaCl concentrations chosen were 0.025, 0.05, and 0.1 M. AOS exhibited electrostatic binding to the positively charged sites of the polypeptide chain resulting in considerable reduction in its hydrodynamic radius up to critical micellar concentration (cmc = 8 mM for no salt, 0.01 and 0.025 M, and 5 mM for 0.05 M and 2 mM for 0.1 M solutions). The correlation function revealed the presence of two types of structures above cmc; namely the micelles of AOS and gelatin-AOS micelle complexes. The micellar radii (Rm), the effective gelatin-surfactant complex radii (Rc), have been determined as a function of salt concentration. No critical aggregation concentration (cac) was observed. The inter-gelatin-surfactant complex (kD1) and inter-micellar interactions (kD2), were determined by fitting the concentration dependence of Rm and Rc to a virial expansion in reduced concentration (c - cmc), which are compared. While kD1 showed strong ionic strength dependence, kD2 remained invariant of the same. The protein to surfactant binding ratio was found to be smaller than normal. Results have been discussed within the framework of the necklace-bead model of polymer-surfactant interactions.     

Amyloid Fibrillation of Insulin under Water-Limited Conditions

Amyloid fibrillation in water-organic mixtures has been widely studied to understand the effect of protein-solvent interactions on the fibrillation process. In this study, we monitored insulin fibrillation in formamide and its methyl derivatives (formamide, N-methyl formamide, N,N-dimethyl formamide) in the presence and absence of water. These model solvent systems mimic the cellular environment by providing denaturing conditions and a hydrophobic environment with limited water content. Thioflavin T (ThT) assay revealed that binary mixtures of water with formamide and its methyl derivatives enhanced fibrillation rates and β-sheet abundance, whereas organic solvents suppressed insulin fibrillation. We utilized solution small-angle x-ray scattering (SAXS) and differential scanning calorimetry (DSC) to investigate the correlation between protein-solvent interactions and insulin fibrillation. SAXS experiments combined with simulated annealing of the protein indicated that the degree of denaturation of the hydrophobic core region at residues B11–B17 determines the fibrillation rate. In addition, DSC experiments suggested a crucial role of hydrophobic interactions in the fibrillation process. These results imply that an environment with limited water, which imitates a lipid membrane system, accelerates protein denaturation and the formation of intermolecular hydrophobic interactions during amyloid fibrillation.     

Local anesthetic-induced microscopic and mesoscopic effects in micelles. A fluorescence,spin label and SAXS study

The interaction of the local anesthetic tetracaine (TTC) with anionic sodium lauryl sulfate (SLS) and zwitterionic 3-(N-hexadecyl-N,N-dimethylammonio)propanesulfonate (HPS) micelles was investigated by fluorescence, spin labeling EPR and small angle X-ray scattering (SAXS). Fluorescence pH titrations allowed the choice of adequate pHs for the EPR and SAXS experiments, where either charged or uncharged TTC would be present. The data also indicated that the anesthetic is located in a less polar environment than its charged counterpart in both micellar systems. EPR spectra evidenced that both anesthetic forms increased molecular organization within the SLS micelle, the cationic form exerting a more pronounced effect. The SAXS data showed that protonated TTC causes an increase in the SLS polar shell thickness, hydration number, and aggregation number, whereas the micellar features are not altered upon incorporation of the uncharged drug. The combined results suggest that the electrostatic interaction between charged TTC and SLS, and the intercalation of the drug in the micellar polar region induce a change in molecular packing with a decrease in the mean cross-sectional area, not observed when the neutral drug sinks more deeply into the micellar hydrophobic domain. In the case of HPS micelles, the EPR spectral changes were small for the charged anesthetic and the SAXS data did not evidence any change in micellar structure, suggesting that this species protrudes more into the aqueous phase due to the lack of electrostatic attractive forces in this system.     

The action of a binary nonionic detergent on a kidney membrane fraction.

The disruption of a kidney cortex microsomal membrane preparation by a binary, nonionic detergent, was followed by using as markers, the changes in total protein content, and (Na+, K+)-ATPase in a supernatant fraction. Both markers responded similarly to changes in pH, microsome concentration and detergent concentration, but responded differently for time-dependent studies. The (Na+, K+)-ATPase activity was increased 2.2-fold (76.1 mumoles Pi/mg protein/h, 95% ouabain-sensitive) by a single detergent treatment and 3.5-fold (92% ouabain-sensitive) by a sequential detergent treatment. Changes in the critical micelle concentration (cmc) were observed for varying detergent and protein concentrations, which suggest interactions of monomeric detergent with the membrane. The peak of (Na+, K+)-ATPase activity occurred above the cmc which suggests the participation of micelles in releasing the enzyme from the membranes. Hill plots of the protein released as the detergent concentration was varied showed a change in the slope near the cmc indicating a four-fold increase in the binding of detergent to membranes as the detergent concentration is increased above the cmc. These results suggest that the disruption of membranes by detergent involves the binding of detergent monomers to the membrane followed by the formation of co-micelles of the detergent with segments of the membrane to complete the separation process.     

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC¹]-Gm; [TOAC¹, Ser^2,6,11,15]-Gm; [Trp⁷]-Gm; [Ser^2,6,11,15, Trp⁷]-Gm; [Trp⁹]-Gm; and [Ser^2,6,11,15, Trp⁹]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a β-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC¹]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.