Lipid Membrane Interface Research Articles

Hydration Dynamics as a Molecular Ruler for Refining Protein Structure at Lipid Membrane Interfaces

Although the 3D protein structure provides the molecular basis for unraveling its unique function, the structural characterization of protein at lipid membrane interfaces is experimentally challenging under physiological conditions. Here, we demonstrate the feasibility of using the long-range and distinct gradient in hydration dynamics along the bilayer normal as a molecular ruler to determine the secondary structure, orientation, and immersion depth of a membrane-associating protein. It was made possible through the quantification of translational diffusion of loosely bound hydration water at the specific sites of protein and lipid bilayer using our newly developed method, Overhauser Dynamic Nuclear Polarization (ODNP)-1H NMR spectroscopy. This study is demonstrated on α-synuclein (αS), a protein associated with Parkinson's disease, free in solution vs. bound on an overall negatively charged phospholipid vesicle. ODNP exhibits that the membrane-bound αS forms an α-helix from residue-76 to 90, where the center of the α-helix is positioned ∼1 below the lipid phosphate. Our data extends the current understanding by showing that residues 90 to 96 of bound αS form a transition segment that links the α-helical domain and the C-terminus with a larger turn than an idealized α-helix, and that the unstructured C-terminus gradually threads outward through a distinct hydration dynamics gradient spanning 10-20 between the water-membrane interface and bulk solvent. Remarkably, ODNP enables to resolve the structural nature of protein functional domains residing 10-20 above the lipid phosphate, which it is difficult to access experimentally. This study debuts the hydration dynamics gradient at protein-lipid interfaces as a high-resolution molecular ruler for probing the structure of membrane proteins and peptides, whose membrane-bound conformation, location and hydration signature are critical to understanding their biological functions.

Disordering of phospholipid headgroups induced by a small amount of polyethylene oxide

We present a (31)P NMR spectroscopy study of planar glass-plate-oriented multi-bilayers of dimyristoylphosphatidylcholine (DMPC) with addition of polyethylene oxide (PEO). This work revealed the presence of a new component in the spectra that appeared only with addition of a small fraction of PEO (up to one PEO segment per dimyristoylphosphatidylcholine molecule) and disappeared when larger amounts of PEO were added. We explained this phenomenon as an effect of an inhomogeneous force field induced by the PEO molecules located at a certain depth in the lipid membrane interface region.

Voltage dependent closure of PorB class II porin from Neisseria meningitidis investigated using impedance spectroscopy in a tethered bilayer lipid membrane interface

Electrochemical impedance spectroscopy (EIS) was used to characterize voltage-dependent closure of PorB class II (PorBII) porin from Neisseria meningitidis incorporated in a tethered bilayer lipid membrane (tBLM). The tBLM’s lower leaflet was fabricated by depositing a self assembled monolayer (SAM) of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) on a gold electrode, and the upper leaflet was formed by depositing1,2-dioleoyl-sn-glycero-3-phoshocholine (DOPC) liposomes. At 0mV bias DC potential, incorporation of PorBII decreased the membrane resistance (Rm) from 2.5MΩcm2 to 0.6MΩcm2, giving a ΔRm of 1.9MΩcm2 and a normalized ΔRm (ΔRm divided by the Rm of the tBLM without PorBII) of 76%. When the bias DC potential was increased to 200mV, the normalized ΔRm value decreased to 20%. The effect of applied voltage on ΔRm was completely reversible, suggesting voltage-dependent closure of PorBII. The voltage dependence of PorBII was further studied in a planar bilayer lipid membrane made from 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhytPC). Following a single insertion event, PorBII exhibited multiple conductance states, with reversible, voltage-dependent closure of PorBII porin occurring at high transmembrane potentials. The trimetric porin closed in three discrete steps, each step corresponding to closure of one conducting monomer unit. The most probable single channel conductance was 4.2nS. The agreement between results obtained with the tBLM and pBLM platforms demonstrates the utility of EIS to screen channel proteins immobilized in tBLM for voltage-gated behavior.

Well-defined lipid interfaces for protein adsorption studies

The biomolecule-artificial lipid membrane interface has been investigated by QCM-D, SPR, and FRAP techniques, to study the adsorption process of ferritin on supported lipid bilayers (SLBs) of different composition and charge. Results point out to the predominant role of electrostatics in triggering the interaction of ferritin with SLBs.

StarD7 behaves as a fusogenic protein in model and cell membrane bilayers

StarD7 is a surface active protein, structurally related with the START lipid transport family. So, the present work was aimed at elucidating a potential mechanism of action for StarD7 that could be related to its interaction with a lipid–membrane interface. We applied an assay based on the fluorescence de-quenching of BD-HPC-labeled DMPC–DMPS 4:1 mol/mol SUVs (donor liposomes) induced by the dilution with non-labeled DMPC–DMPS 4:1 mol/mol LUVs (acceptor liposomes). Recombinant StarD7 accelerated the dilution of BD-HPC in a concentration-dependent manner. This result could have been explained by either a bilayer fusion or monomeric transport of the labeled lipid between donor and acceptor liposomes. Further experiments (fluorescence energy transfer between DPH-HPC/BD-HPC, liposome size distribution analysis by dynamic light scattering, and the multinuclear giant cell formation induced by recombinant StarD7) strongly indicated that bilayer fusion was the mechanism responsible for the StarD7-induced lipid dilution. The efficiency of lipid dilution was dependent on StarD7 electrostatic interactions with the lipid–water interface, as shown by the pH- and salt-induced modulation. Moreover, this process was favored by phosphatidylethanolamine which is known to stabilize non-lamellar phases considered as intermediary in the fusion process. Altogether these findings allow postulate StarD7 as a fusogenic protein.

Suppression of IAPP fibrillation at anionic lipid membranes via IAPP-derived amyloid inhibitors and insulin

Aggregation of human islet amyloid polypeptide (hIAPP) into cytotoxic β-sheet oligomers and amyloid plaques is considered a key event in pancreatic β-cell degeneration in type 2 diabetes (T2D). hIAPP is synthesized in the pancreatic β-cells and it is stored, co-processed in the secretory granules, and co-secreted to the extracellular matrix together with insulin. In vivo, hIAPP aggregation may start and proceed at the water-cell membrane interface and anionic lipid membranes strongly enhance the process of hIAPP fibrillization which is causally linked to membrane disintegration and cell degeneration. In this study we explored the amyloidogenic propensity and conformational properties of hIAPP in the presence of negatively charged membrane (DOPC/DOPG phospholipid bilayers) surfaces upon addition of two recently designed potent hIAPP-derived inhibitors of hIAPP amyloidogenesis, the hexapeptide NF(N-Me)GA(N-Me)IL (NFGAIL-GI) and the 37-residue non-amyloidogenic hIAPP analog [(N-Me)G24, (N-Me)I26]-IAPP (IAPP-GI). For comparison, the effects of insulin, which is a natively occurring hIAPP aggregation inhibitor, rat IAPP (rIAPP), which is a natively non-amyloidogenic hIAPP analog, and the hIAPP amyloid core peptide hIAPP(22-27) or NFGAIL were also studied. The aim of our study was to test whether and how the above peptides which have been shown to completely block or suppress hIAPP amyloidogenesis in bulk solution in vitro would also affect these processes in the presence of lipid membranes. To this end, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) was applied. We find that IAPP-GI, NFGAIL-GI, insulin, and rIAPP are potent inhibitors of hIAPP fibrillization. Importantly, our data also suggest that the hetero-complexes of IAPP-GI, rIAPP, and insulin with hIAPP although non-amyloidogenic per se are still able to adsorb at the lipid membrane. By contrast, in the presence of NFGAIL-GI, interaction of hIAPP with the lipid membrane is completely abolished, consistent with NFGAIL-GI mediated sequestration of hIAPP via hetero-complexation in the aqueous phase mainly accounting for the observed strong effect of NFGAIL-GI on hIAPP fibrillogenesis at the lipid membrane interface. Finally, our studies show that once hIAPP is fibrillized at the water-lipid membrane interface with fibrils being attached to the lipid membrane, it cannot be disaggregated by all above peptides.

Lipid-Membrane Mediated Tau Misfolding and Aggregation

Neurofibrillary tangles comprise of aggregated tau protein are a pathological hallmark of Alzheimer's disease (AD). However, the molecular basis of the early tau aggregation events, such as the nature of the structural fluctuations that trigger the cascade of misfolding and aggregation events, are unknown. Several studies suggest that tau in AD brains may exhibit abnormal interactions with the neuronal cell membrane. We hypothesize that the lipid membrane can mediate tau pathology by templating tau to misfold into an assembly-competent conformation and subsequently nucleating tau to aggregate into fibrils. We used lipid monolayers at the air/water interface as a model membrane to probe tau-membrane interactions. We found that although tau (hTau40) is highly soluble and charged, it is also highly surface active. hTau40 exhibits strong association with negative DMPG lipids, while exhibiting weaker interactions with the positive DMTAP and neutral DMPC lipids. Thus, tau-membrane interactions are strongly mediated by electrostatic interactions. To identify the hTau40 domain that is responsible for its interaction with membranes, we measured the interaction between different tau constructs (K18 and K32) and lipid membranes. Additionally, X-ray scattering experiments were carried out to elucidate the structural details of tau associated with lipid membranes. Our data show that tau's C-terminal, microtubule binding domain, is responsible for its association with the lipid membrane and that these binding events disrupts the ordering and structure of the membrane. Our study suggests that the “soft” nature of tau can give rise to rich dynamic behaviors at interfaces, such as the physiological lipid membrane interface. Our data implicate that the inner leaflet of the cell membrane, enriched in negatively charged lipids, can potentially recruit tau in the cytoplasm, which may be critical in initiating the cascade of pathogenic misfolding and aggregation events in AD.

All-atom molecular dynamics simulation studies of fully hydrated gel phase DPPG and DPPE bilayers

Here in silico lipid membranes are described providing a structural background of the organization of the lipid components of membranes and aiding further biological or biophysical studies. An all-atom molecular dynamics simulations has been performed to investigate structural and dynamical properties of two fully hydrated gel-phase bilayers of 1,2-dipalmitoyl- sn-glycero-3-phosphoglycerol (DPPG) and 1,2-dipalmitoyl- sn-glycero-3-phospho-ethanolamine (DPPE) bilayers at 303 K. The respective starting configuration of lipids in the simulation bilayer unit cells were taken on the basis of scattering data. In both simulations, we found overall reasonably good agreement with the available experimental data (area per lipid, phosphorus–phosphorus distance). The distribution of the water/counterions at the membrane interface, interactions/orientations of lipid headgroups, and hydrocarbon chain organization were extensively studied in terms of pair distribution functions between main structural components of the system. Intra/intermolecular hydrogen bond formation was discussed in detail. The water orientation at the lipid membrane interface was explored thoroughly in terms of dipole moment as a function of the water molecule positions along the membrane, where we found that the counterions changed the orientation of the water at the interface. Special attention has been devoted to the distribution of the sodium counterions around the DPPG headgroup. We found preferential binding of Na + ions to the phosphate oxygen species.

Phase Transitions in Phospholipid Monolayers Studied by Atomic Force Microscopy and Langmuir-Blodgett Technique

The phase behaviour and surface structure of dipalmitoyl phosphatidyl choline (DPPC) monolayers at the air/water interface, in the absence and the presence of procaine, have been investigated by Langmuir-Blodgett (LB) technique and atomic force microscopy. The LB films were transferred on mica, at a controlled surface pressure, characteristic for the expanded liquid to condensed liquid phase transition of pure DPPC monolayers. The results indicate that procaine penetrates into and specifically interacts with phospholipid monolayers stabilizing the lipid membrane interface.

Molecular dynamics simulations of microstructure and mixing dynamics of cryoprotective solvents in water and in the presence of a lipid membrane

Molecular dynamics (MD) simulation is used to investigate the solubility behavior of cryoprotective (CP) solvents, such as DMSO, ethylene glycol (EG) and glycerol (GL), in pure water and in the presence of a lipid membrane. The MD study is focused on an equilibration timescale required for mixing large CP aggregates with aqueous and aqueous/lipid environments. The MD analysis demonstrates that DMSO mixes rapidly with water, so that all solute molecules are uniformly distributed in the equilibrium aqueous solution. Our investigation of the microstructure of binary EG/water and GL/water systems reveals that, despite the miscibility of both CP solvents with water, they are not ideally mixed in aqueous solutions at the molecular level. The MD simulations show that the mixing dynamics of the large CP cluster and surrounding water is found to be strongly dependent on nature of hydrophilic and hydrophobic interactions acting between cryoprotectant molecules. In particular, a spatial hydrogen-bond network formed between CP molecules plays an important role in the mixing dynamics between CP agents and water. A further analysis on the mixing behavior of the CP solvents with pure water and with aqueous solutions at a lipid membrane interface shows that, due to strong binding of the CP molecules to membrane surface, the equilibration process in the lipid environment becomes very slow, at least of the order of microseconds. The MD results are discussed in the context of the better understanding on the composition of the aqueous mixtures of the EG and GL solvents. Knowledge of the microstructure and the dynamics of these systems helps to develop better cryopreservation protocols and to propose more optimal cooling/warming regimes for cellular cryosolutions.

Impact of Hapten Presentation on Antibody Binding at Lipid Membrane Interfaces

We report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (KD) were compared for two model systems, biotin/anti-biotin and 2,4-dinitrophenyl (DNP)/anti-DNP, in bulk solution and at model membrane surfaces. The binding constants in solution were obtained from fluorescence anisotropy measurements. The surface binding constants were determined by microfluidic techniques in conjunction with total internal reflection fluorescence microscopy. The results showed that the bulk solution equilibrium dissociation constants for anti-biotin and anti-DNP were almost identical, KD(bulk)=1.7±0.2 nM vs. 2.9±0.1 nM. By contrast, the dissociation constant for anti-biotin antibody was three orders of magnitude tighter than for anti-DNP at a lipid membrane interface, KD=3.6±1.1 nM vs. 2.0±0.2 μM. We postulate that the pronounced difference in surface binding constants for these two similar antibodies is due to differences in the ligands’ relative lipophilicity, i.e., the more hydrophobic DNP molecules had a stronger interaction with the lipid bilayers, rendering them less available to incoming anti-DNP antibodies compared with the biotin/anti-biotin system. However, when membrane-bound biotin ligands were well screened by a poly(ethylene glycol) (PEG) polymer brush, the KD value for the anti-biotin antibody could also be weakened by three orders of magnitude, 2.4±1.1μM. On the other hand, the dissociation constant for anti-DNP antibodies at a lipid interface could be significantly enhanced when DNP haptens were tethered to the end of very long hydrophilic PEG lipopolymers (KD=21±10nM) rather than presented on short lipid-conjugated tethers. These results demonstrate that ligand presentation strongly influences protein interactions with membrane-bound ligands.

Binding and interactions of L-BABP to lipid membranes studied by molecular dynamic simulations

Chicken liver bile acid-binding protein (L-BABP) is a member of the fatty acid-binding proteins super family. The common fold is a β-barrel of ten strands capped with a short helix–loop–helix motif called portal region, which is involved in the uptake and release of non-polar ligands. Using multiple-run molecular dynamics simulations we studied the interactions of L-BABP with lipid membranes of anionic and zwitterionic phospholipids. The simulations were in agreement with our experimental observations regarding the electrostatic nature of the binding and the conformational changes of the protein in the membrane. We observed that L-BABP migrated from the initial position in the aqueous bulk phase to the interface of anionic lipid membranes and established contacts with the head groups of phospholipids through the side of the barrel that is opposite to the portal region. The conformational changes in the protein occurred simultaneously with the binding to the membrane. Remarkably, these conformational changes were observed in the portal region which is opposite to the zone where the protein binds directly to the lipids. The protein was oriented with its macrodipole aligned in the configuration of lowest energy within the electric field of the anionic membrane, which indicates the importance of the electrostatic interactions to determine the preferred orientation of the protein. We also identified this electric field as the driving force for the conformational change. For all the members of the fatty acid-binding protein family, the interactions with lipid membranes is a relevant process closely related to the uptake, release and transfer of the ligand. The observations presented here suggest that the ligand transfer might not necessarily occur through the domain that directly interacts with the lipid membrane. The interactions with the membrane electric field that determine orientation and conformational changes described here can also be relevant for other peripheral proteins.

Tryptophan orientation in model lipid membranes

Tryptophans in membrane proteins display strong preference for the lipid membrane interface and are important for anchoring proteins at the proper longitudinal level. Linear dichroism spectroscopy on indoles in shear-deformed liposomes has been used to show that this positioning is accompanied by an intrinsically adopted orientation, also observed for tryptophans in membrane-bound peptides. Similarities in orientation of different indoles suggest that tryptophan will adopt this orientation independent of the protein it is part of. From the orientation of indole electronic transition moments L a, L b and B b, a binding model is proposed where the indole long axis is ∼60–65° from the membrane normal and the indole plane is at an oblique angle. We propose that dipole–dipole interactions and steric constraints in the membrane hydrocarbon region determine positioning and orientation of tryptophans whereas hydrogen bonding and cation-π interactions with lipid head-groups, though contributing to the membrane affinity of indoles, are less important.

The effect of peptides and ions interacting with an electrically neutral membrane interfaceon the structure and stability of lipid membranes in the liquid-crystalline phase and in theliquid-ordered phase

We investigated the effects of a de novo designed peptide, WLFLLKKK (peptide-1) andLa3+, which can bind with the electrically neutral lipid membrane interface,on the stability of the phosphatidylcholine (PC) membrane in theLα phase and that of the liquid-ordered (lo) phase membranes. The results of spacing of themultilamellar vesicle and shape changes of the giant unilamellar vesicle (GUV)indicate that the peptide-1 can be partitioned into the membrane interface in theLα phase but not intothat in the lo phase. La3+ induced shape changes of GUVs of the lo phase membrane, which are the same as those of GUVs in theLα phase. This indicatesthat the binding of La3+ induced an increase in the lateral compression pressure of the membrane, which decreased thesurface area of the membrane in the lo phase. The difference of the membrane interface between theLα phase and the lo phase is discussed.

Surface activity and interaction of StarD7 with phospholipid monolayers

StarD7 protein forms stable Gibbs and Langmuir monolayers at the air–buffer interface showing marked surface activity. The latter is enhanced by penetration into phospholipid films at an initial surface pressure above the protein’s own equilibrium adsorption surface pressure to a lipid-free interface. The protein–phospholipid stabilizing interactions at the interface depend on the lipid, with preference for phosphatidylserine, cholesterol, and phosphatidylglycerol, and the increases of lateral surface pressure generated are comparable to those of other membrane-active proteins. The surface activity of StarD7 is strong enough to thermodynamically drive and retain StarD7 at the lipid membrane interface where it may undergo lipid-dependent reorganization as indicated by changes of surface pressure and electrostatics.

Effect of de Novo Designed Peptides Interacting with the Lipid-Membrane Interface on the Stability of the Cubic Phases of the Monoolein Membrane

Elucidation of the mechanism of transitions between the cubic phase and the liquid-crystalline (Lα) phase and between different cubic phases is essential for an understanding of the dynamics of biomembranes and the development of new crystallization techniques. Recently, we found that electrostatic interactions due to the surface charges of lipid membranes induce transitions between the cubic phase and Lα phase and between different cubic phases (Aota-Nakano, Y.; Li, S. J.; Yamazaki, M. Biochim. Biophys. Acta 1999, 1461, 96; Li, S. J.; Yamashita, Y.; Yamazaki, M. Biophys. J. 2001, 81, 983). In the present study, we used small-angle X-ray scattering to investigate the effects of a de novo designed peptide (WLFLLKKK, peptide-1, which has positive charges and a site that is partitioned in electrically neutral lipid-membrane interfaces) on the stability of the cubic phase of the monoolein (MO) membrane. As the peptide-1 concentration increased, a transition from the Q224 to the Q229 phase in the MO membrane at a 30 wt % lipid concentration occurred at R = 0.0090 (R is the molar ratio of peptide to MO), and at R ≥ 0.040, the MO/peptide-1 membranes were in the Lα phase. As the NaCl concentration in the bulk phase increased, for the MO/peptide-1 membranes in the Q229 phase in excess water, a Q229 phase to Q224 phase transition occurred at low concentrations of NaCl. Similarly, for the MO/peptide-1 membranes (R = 0.080) in excess water, at low NaCl concentrations, they were in the Lα phase, but at ≥0.40 M NaCl, they were in the Q224 phase. These results indicate that peptide-1 was partitioned in the membrane interface of the MO membrane, electrostatic interactions due to peptide-1 in the membrane interface make the Q229 phase more stable than the Q224 phase, and with larger electrostatic interactions, the Lα phase is more stable than these cubic phases. Increased peptide-1 concentration reduced the absolute value of spontaneous curvature of the MO/peptide-1 monolayer membrane. On the basis of these results, we discuss the mechanism of the effect of peptide-1 on the phase stability of the MO membranes.

Shape Changes of Giant Unilamellar Vesicles of Phosphatidylcholine Induced by a De Novo Designed Peptide Interacting with Their Membrane Interface

We have designed and synthesized a peptide, WLFLLKKK (peptide-1), which has positive charges and a segment partitioned into the electrically neutral lipid membrane interface, and investigated its effect on the stability of the phosphatidylcholine (PC) membrane. Spacing of multilamellar vesicles of palmitoyloleoyl-PC increased greatly with an increase in peptide-1 concentration. The addition of 5 μM peptide-1 through a micropipet near the giant unilamellar vesicle (GUV) of dioleoylphosphatidylcholine induced several kinds of shape changes; for example, a discocyte was changed to two spheres connected by a neck, and small vesicles were budded into the outside of the spherical GUV. These results indicate that the de novo designed peptide-1 can be partitioned into the PC membrane interface and have a large effect on its structure and properties.

Fluoroquinolone−Biomembrane Interaction at the DPPC/PG Lipid−Bilayer Interface

The interaction of two fluoroquinolones, ciprofloxacin (CPX) and its N-4-butylpiperazinyl derivative (BCPX), with liposomes bearing negative surface charge was studied by fluorescence methods. Binding of 1-anilino-8-naphthalenesulfonate to the liposome surface and diphenylhexatriene (DPH) and diphenylhexatriene−propionic (DPH−AP) acid fluorescence anisotropy measurements were differently affected by the two. On the basis of the variations of the surface potential and phase transition temperature (Tm) of the phospholipids studied, it was concluded that (i) both drugs interact electrostatically at the lipid membrane interface and (ii) the butyl side chain of BCPX led to a different interaction with the lipid headgroups. This behavior is then related to the activity of an efflux pump Serratia marcescens NIMA, which appear to be affected by the ability of CPX and BCPX to interact with the inner surface of the plasma membrane.

1G1600 ペプチドが誘起する生体膜のキュービック相から2分子層膜への相転移(13.生体膜・人工膜(A)構造・物性,一般演題,日本生物物理学会第40回年会)

1G1600 ペプチドが誘起する生体膜のキュービック相から2分子層膜への相転移(13.生体膜・人工膜(A)構造・物性,一般演題,日本生物物理学会第40回年会)

Candidacidal activity prompted by N-terminus histatin-like domain of human salivary mucin (MUC7)

Histidine-rich peptides (histatins, Hsn) in saliva are thought to provide a non-immune defense against Candida albicans. Sequence homology search of the human salivary mucin, MUC7, against histatins revealed a domain at the N-terminus (R 3–Q 17) having 53% identity to Hsn-5. To determine its candidacidal activity, this 15 residue basic histidine-rich domain of MUC7 ( I) was prepared by solid-phase Fmoc chemistry. Various N- and C-terminal protected derivatives of I were also synthesized to correlate the effect of peptide overall charge in exhibiting cidal potency. Candidacidal activity measurement of I and its variants showed considerable ED 50 values (effective dosage required to kill 50% of candida cells), albeit greater than Hsn-5 (ED 50 ∼4–6 μM). Of the various analogs tested, N-terminal free acid ( I, ED 50 ∼40 μM) and amide ( V, ED 50∼16 μM) exhibited appreciable candidacidal activities suggesting the possible role of peptide net charge in cidal action. Blocking of N-terminus with a bulky octanoyl group showed only marginal effect on the cidal activity of I or V, indicating that hydrophobicity of these synthetic constructs may not be important for exerting such activities. Membrane-induced conformational transition from random coil to helical structures of all the test peptides implied their tendency to adapt order structures at the lipid-membrane interface similar to that of Hsn-5. However, comparison of propensity for helical structure formation vs. ED 50 indicated that cidal potency of MUC7 Hsn-like peptides depends largely on electrostatic interactions irrespective of secondary structural elements. Delineation of solution structure of the most active peptide ( V) by 2D-NMR revealed essentially a non-structured conformation in aqueous medium, which further supported the fact that the peptide helical structure may not be a prerequisite for posing candidacidal activity. The formation of smaller truncated peptides and/or Hsn-like fragments on proteolytic degradation of intact MUC7 in the presence of oral flora provided indirect evidence that mucin could serve as a backup candidacidal agent to salivary Hsn.