Polyphosphoinositides (PPIs) and in particular phosphatidylinositol-(4,5)-bisphosphate (PI4,5P2), control many cellular events and bind with variable levels of specificity to hundreds of intracellular proteins in vitro. The much more restricted targeting of proteins to PPIs in cell membranes is thought to result in part from the formation of spatially distinct PIP2 pools, but the mechanisms that cause formation and maintenance of PIP2 clusters are still under debate. The hypothesis that PIP2 forms sub-micron-size clusters in the membrane by electrostatic interactions with intracellular divalent cations is tested here using lipid monolayer and bilayer model membranes. Competitive binding between Ca2+ and Mg2+ to PIP2 is quantified by surface pressure measurements and analyzed by a Langmuir competitive adsorption model. The physical chemical differences among three PIP2 isomers are also investigated. Addition of Ca2+, but not Mg2+, Zn2+ or polyamines to PIP2-containing monolayers induces surface pressure drops coincident with the formation of PIP2 clusters visualized by fluorescence, atomic force and electronic microscopy. Studies of bilayer membranes using steady-state probe-partitioning fluorescence resonance energy transfer (SP-FRET) and fluorescence correlation spectroscopy (FCS) also reveal Me2+-induced cluster formation or diffusion retardation which follows the trend: Ca2+ ≫ Mg2+ > Zn2+, and polyamines have minimal effects. These results suggest that divalent metal ions have substantial effects on PIP2 lateral organization at physiological concentrations, and local fluxes in their cytoplasmic levels can contribute to regulating protein-PIP2 interactions.

Many antimicrobial peptides (AMPs) function by forming various oligomeric structures and/or pores upon binding to bacterial membranes. Because such peptide aggregates are capable of inducing membrane thinning and membrane permeabilization, it is expected that AMP-binding would also affect the diffusivity or mobility of the lipid molecules in the membrane. Herein, we show that measurements of the diffusion times of individual lipids through a confocal volume via fluorescence correlation spectroscopy (FCS) provide a sensitive means to probe the underlying AMP-membrane interactions. In particular, results obtained with two well-studied AMPs, magainin 2 and mastoparan X, and two model membranes indicate that this method is capable of revealing structural information, especially the heterogeneity of the peptide-membrane system, that is otherwise difficult to obtain using common ensemble methods. Moreover, because of the high sensitivity of FCS, this method allows examination of the effect of AMPs on the membrane structure at very low peptide/lipid ratios.

The design of β-peptide foldamers targeting the transmembrane (TM) domains of complex natural membrane proteins has been a formidable challenge. A series of β-peptides was designed to stably insert in TM orientations in phospholipid bilayers. Their secondary structures and orientation in the phospholipid bilayer was characterized using biophysical methods. Computational methods were then devised to design a β-peptide that targeted a TM helix of the integrin αIIbβ3. The designed peptide (β-CHAMP) interacts with the isolated target TM domain of the protein, and activates the intact integrin in vitro.

Preterm birth is the leading cause of neonatal mortality, and is frequently associated with intra-amniotic infection hypothesized to arise from bacterial ascension across a dysfunctional cervical mucus plug. To study this dysfunction, we assessed the permeability of cervical mucus from non-pregnant ovulating (n = 20) and high- (n = 9) and low-risk (n = 16) pregnant women to probes of varying sizes and surface chemistries. We found that the motion of negatively charged, carboxylated microspheres in mucus from pregnant patients was significantly restricted compared to ovulating patients, but not significantly different between high- and low-risk pregnant women. In contrast, charged peptide probes small enough to avoid steric interactions, but sensitive to the biochemical modifications of mucus components exhibited significantly different transport profiles through mucus from high- and low-risk patients. Thus, although both microstructural rearrangements of the components of mucus as well as biochemical modifications to their adhesiveness may alter the overall permeability of the cervical mucus plug, our findings suggest that the latter mechanism plays a dominant role in the impairment of the function of this barrier during preterm birth. We expect that these probes may be readily adapted to study the mechanisms underlying disease progression on all mucosal epithelia, including those in the mouth, lungs, and gut.

Recently we have shown that association with an antimicrobial peptide (AMP) can drastically alter the diffusion behavior of the constituent lipids in model membranes (Biochemistry 49,(4672)(4673)(4674)(4675)(4676)(4677)(4678). In particular, we found that the diffusion time of a tracer fluorescent lipid through a confocal volume measured via fluorescence correlation spectroscopy (FCS) is distributed over a wide range of timescales, indicating the formation of stable and/or transient membrane species that have different mobilities. A simple estimate, however, suggested that the slow diffusing species are too large to be attributed to AMP oligomers or pores that are tightly bound to a small number of lipids. Thus, we tentatively ascribed them to membrane domains and/or clusters that possess distinctively different diffusion properties. In order to further substantiate our previous conjecture, herein we study the diffusion behavior of the membrane-bound peptide molecules using the same AMPs and model membranes. Our results show, in contrast to our previous findings, that the diffusion times of the membrane-bound peptides exhibit a much narrower distribution that is more similar to that of the lipids in peptide-free membranes. Thus, taken together, these results indicate that while AMP molecules prompt domain formation in membranes, they are not tightly associated with the lipid domains thus formed. Instead, they are likely located at the boundary regions separating various domains and acting as mobile fences. KeywordsMembrane; Antimicrobial Peptide; Fluorescence Correlation Spectroscopy; Diffusion; Membrane Domain FormationThe mechanism of action of antimicrobial peptides (AMPs) has been the subject of extensive studies (1-7). Findings from these studies have prompted the formulation of several models of membrane disruption by AMPs. For example, the ability of AMPs to disrupt the structural integrity of the targeted cell membranes has been attributed to (a) barrel-stave or/and toroidal pore formation (8,9), (b) membrane-dissolution in a detergentlike manner (10,11), (c) formation of lipid-peptide domains (12-25), (d) segregation of anionic lipids and zwitterionic lipids (23-27), or (e) formation of non-lamellar phases (28-30). Recently, we have shown that the diffusivity of individual lipids in an AMP-bound membrane, probed via fluorescence correlation spectroscopy (FCS), provides a sensitive means to monitor how AMP binding affects the membrane's structure and dynamics (31), † Supported by the NIH (GM-065978) and the NSF (DMR05-20020).* To whom correspondence should be addressed; gai@sas.upenn.edu; Phone: 215-573-6256; Fax: 215-573-2112 even at very low peptide/lipid ratios. As shown (Figure 1), results obtained from two wellstudied AMPs, magainin 2 (mag2) and mastoparan X (mpX), showed that AMP-binding can drastically alter the lipid diffusion behavior, and that at relatively high peptide/lipid ratios the lipid diffusion times through a well defined confocal volume, acquired by repeating measurements, are...