Three structural roles for water in bone observed by solid-state NMR

Hydrogen-bearing species in the bone mineral environment were investigated using solid-state NMR spectroscopy of powdered bone, deproteinated bone, and B-type carbonated apatite. Using magic-angle spinning and cross-polarization techniques three types of structurally-bound water were observed in these materials. Two of these water types occupy vacancies within the apatitic mineral crystal in synthetic carbonated apatite and deproteinated bone and serve to stabilize these defect-containing crystals. The third water was observed at the mineral surface in unmodified bone but not in deproteinated bone, suggesting a role for this water in mediating mineral-organic matrix interactions. Direct evidence of monohydrogen phosphate in a ¹H NMR spectrum of unmodified bone is presented for the first time. We obtained clear evidence for the presence of hydroxide ion in deproteinated bone by ¹H MAS NMR. A ¹H-³¹P heteronuclear correlation experiment provided unambiguous evidence for hydroxide ion in unmodified bone as well. Hydroxide ion in both unmodified and deproteinated bone mineral was found to participate in hydrogen bonding with neighboring water molecules and ions. In unmodified bone mineral hydroxide ion was found, through a ¹H-³¹P heteronuclear correlation experiment, to be confined to a small portion of the mineral crystal, probably the internal portion.     

Solid-phase 31P NMR spectra of peat and mineral soils,humic acids and soil solution components: influence of iron and manganese

Solid-phase³¹P nuclear magnetic resonance (NMR) offers a direct means to determine the chemical environment of P present in soil and soil fractions. Iron is often a major component in soil and it has been thought that the presence of paramagnetic Fe and Mn in soil components is responsible for loss of resolution in NMR spectra. We have found that the resolution of signals in the solid-phase ³¹P NMR spectra of a Fe- and Mn-rich mineral soil was no worse than that for a series of four peat soils with a comparable concentration of P. Similarly, the resolution in the solid-phase ³¹P NMR spectra of the humic acid from the mineral soil was not much changed by extraction of the humic acid with acetylacetone in diethyl ether which removed around 40% of its Fe and 30% of its Mn. Removal of up to 50% of the Fe from organic rich, freeze-dried soil solutions from a soil catena with different land uses produced little change in spectral resolution. It was concluded that the limitations to resolution in solid-phase ³¹P NMR spectroscopy of soil humic substances do not stem from the presence of paramagnetic substances, but from the variable way P species are physically held in the amorphous milieu of the organic phase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Unique backbone-water interaction detected in sphingomyelin bilayers with 1H/31P and 1H/13C HETCOR MAS NMR spectroscopy

Two-dimensional ¹H/³¹P dipolar heteronuclear correlation (HETCOR) magic-angle spinning nuclear magnetic resonance (NMR) is used to investigate the correlation of the lipid headgroup with various intra- and intermolecular proton environments. Cross-polarization NMR techniques involving ³¹P have not been previously pursued to a great extent in lipid bilayers due to the long ¹H-³¹P distances and high degree of headgroup mobility that averages the dipolar coupling in the liquid crystalline phase. The results presented herein show that this approach is very promising and yields information not readily available with other experimental methods. Of particular interest is the detection of a unique lipid backbone-water intermolecular interaction in egg sphingomyelin (SM) that is not observed in lipids with glycerol backbones like phosphatidylcholines. This backbone-water interaction in SM is probed when a mixing period allowing magnetization exchange between different ¹H environments via the nuclear Overhauser effect (NOE) is included in the NMR pulse sequence. The molecular information provided by these ¹H/³¹P dipolar HETCOR experiments with NOE mixing differ from those previously obtained by conventional NOE spectroscopy and heteronuclear NOE spectroscopy NMR experiments. In addition, two-dimensional ¹H/¹³C INEPT HETCOR experiments with NOE mixing support the ¹H/³¹P dipolar HETCOR results and confirm the presence of a H₂O environment that has nonvanishing dipolar interactions with the SM backbone.     

Autoradiographic localization of technetium-99 methylene diphosphonate in growth sites of young mice

Conflicting reports about the localization of ^99mTc-MDP in bone and cartilage are found in the literature. Possible binding sites include hydroxyapatite and non-osteoid matrix such as immature collagen. The present study used autoradiographs of demineralized and non-demineralized growth sites in young mice to demonstrate localization of ⁹⁹Tc-MDP, and consequently ^99mTc-MDP, in mineralizing cartilage and bone. Uptake of the isotope was seen in mineralizing bone and cartilage, associated with the mineral in contrast to the organic phase. The results indicate that bone seeking radiopharmaceutical uptake (BSRU) may detect alterations in the rate of mineralized phase in growth sites and thus has the potential to disclose skeletal growth disorders.     

Control of Vertebrate Skeletal Mineralization by Polyphosphates

Background

Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO₃ ⁻)_n) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.

Principal Findings/Methodology

The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO₄ ³⁻) concentration while permitting the accumulation of a high total PO₄ ³⁻ concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO₄ ³⁻ and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4′,6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation.

Conclusions/Significance

We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.     

The unique alternation of conformationally different (‘chair’-‘saddle’) eight-membered metallocycles [Cd2S4P2] in the chains of cadmium dialkyldithiophosphates: C, P, Cd CP/MAS NMR and single-crystal X-ray diffraction studies

O,O′-Dipropyldithiophosphate and O,O′-dibutyldithiophosphate (Dtph) cadmium(II) complexes were prepared and studied by means of heteronuclear ³¹P, ¹¹³Cd, ³¹C CP/MAS NMR spectroscopy and single-crystal X-ray diffraction. Linear-chain polynuclear structures have been established for both cadmium(II) complexes, in which each pair of equivalent dithiophosphate groups, playing the same bridging structural function, asymmetrically links the neighbouring cadmium atoms. One remarkable structural feature of the synthesised cadmium(II) compounds is defined by the alternation of two types of conformationally different (‘chair’-‘saddle’) eight-membered rings [Cd₂S₄P₂] in the polymeric chains. Therefore, in both ³¹P NMR and XRD data, the bridging dithiophosphate ligands exhibit structural inequivalence in pairs. The structural states of both Dtph ligands and cadmium atoms have been characterised by the ³¹P and ¹¹³Cd chemical shift tensors, which display a profound axially symmetric and mainly rhombic characters, respectively. All experimental ³¹P resonances were assigned to the phosphorus structural sites in both resolved structures.     

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method

Summary The ultrastructural localization of alkaline phosphatase (AlP) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl₂ to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl₂ and CeCl₃. AlP activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.     

Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds

The most widely accepted hypothesis to account for maturational changes in the X-ray diffraction characteristics of bone mineral has been the 'amorphous calcium phosphate theory', which postulates that an initial amorphous calcium phosphate solid phase is deposited that gradually converts to poorly crystalline hydroxyapatite. Our studies of bone mineral of different ages by X-ray radial distribution function analysis and 31P n.m.r. have conclusively demonstrated that a solid phase of amorphous calcium phosphate does not exist in bone in any significant amount. 31P n.m.r. studies have detected the presence of acid phosphate groups in a brushite-like configuration. Phosphoproteins containing O-phosphoserine and O-phosphothreonine have been isolated from bone matrix and characterized. Tissue and cell culture have established that they are synthesized in bone, most likely by the osteoblasts. Physiochemical and pathophysiological studies support the thesis that the mineral and organic phases of bone and other vertebrate mineralized tissues are linked by the phosphomonester bonds of O-phosphoserine and O-phosphothreonine, which are constituents of both the structural organic matrix and the inorganic calcium phosphate crystals.     

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Diffusion of Exchangeable Water in Cortical Bone Studied by Nuclear Magnetic Resonance

The rate-limiting step in the delivery of nutrients to osteocytes and the removal of cellular waste products is likely diffusion. The transport of osteoid water across the mineralized matrix of bone was studied by proton nuclear magnetic resonance spectroscopy and imaging by measuring the diffusion fluxes of tissue water in cortical bone specimens from the midshaft of rabbit tibiae immersed in deuterium oxide. From the diffusion coefficient (D_a = (7.8 ± 1.5) × 10⁻⁷ cm²/s) measured at 40°C (close to physiological temperature), it can be inferred that diffusive transport of small molecules from the bone vascular system to the osteocytes occurs within minutes. The activation energy for water diffusion, calculated from D_a measured at four different temperatures, suggests that the interactions between water molecules and matrix pores present significant energy barriers to diffusion. The spatially resolved profile of D_a perpendicular to the cortical surface of the tibia, obtained using a finite difference model, indicates that diffusion rates are higher close to the endosteal and periosteal surfaces, decreasing toward the center of the cortex. Finally, the data reveal a water component (∼30%) diffusing four orders of magnitude more slowly, which is ascribed to water tightly bound to the organic matrix and mineral phase.     

Evidence of pores and thinned lipid bilayers induced in oriented lipid membranes interacting with the antimicrobial peptides, magainin-2 and aurein-3.3

Dynamic structures of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers induced in oriented lipid membranes, which are interacting with membrane-acting antimicrobial peptides (AMPs), magainin-2 and aurein-3.3, were explored by ³¹P and ²H solid-state NMR (ssNMR) spectroscopy. Various types of phospholipid systems, such as POPC-d₃₁, POPC-d₃₁/POPG, and POPC-d₃₁/cholesterol, were investigated to understand the membrane disruption mechanisms of magainin-2 and aurein-3.3 peptides at various peptide-to-lipid (P:L) ratios. The experimental lineshapes of anisotropic ³¹P and ²H ssNMR spectra measured on these peptide-lipid systems were simulated reasonably well by assuming the presence of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers, in membranes. Furthermore, the observed decrease in the anisotropic frequency span of either ³¹P or ²H ssNMR spectra of oriented lipid bilayers, particularly when anionic POPG lipids are interacting with AMPs at high P:L ratios, can directly be explained by a thinned membrane surface model with fast lateral diffusive motions of lipids. The spectral analysis protocol we developed enables extraction of the lateral diffusion coefficients of lipids distributed on the curved surfaces of pores and thinned bilayers on a few nanometers scale.     

Total Water,Phosphorus Relaxation and Inter-Atomic Organic to Inorganic Interface Are New Determinants of Trabecular Bone Integrity

Bone is the living composite biomaterial having unique structural property. Presently, there is a considerable gap in our understanding of bone structure and composition in the native state, particularly with respect to the trabecular bone, which is metabolically more active than cortical bones, and is readily lost in post-menopausal osteoporosis. We used solid-state nuclear magnetic resonance (NMR) to compare trabecular bone structure and composition in the native state between normal, bone loss and bone restoration conditions in rat. Trabecular osteopenia was induced by lactation as well as prolonged estrogen deficiency (bilateral ovariectomy, Ovx). Ovx rats with established osteopenia were administered with PTH (parathyroid hormone, trabecular restoration group), and restoration was allowed to become comparable to sham Ovx (control) group using bone mineral density (BMD) and µCT determinants. We used a technique combining ¹H NMR spectroscopy with ³¹P and ¹³C to measure various NMR parameters described below. Our results revealed that trabecular bones had diminished total water content, inorganic phosphorus NMR relaxation time (T₁) and space between the collagen and inorganic phosphorus in the osteopenic groups compared to control, and these changes were significantly reversed in the bone restoration group. Remarkably, bound water was decreased in both osteopenic and bone restoration groups compared to control. Total water and T₁ correlated strongly with trabecular bone density, volume, thickness, connectivity, spacing and resistance to compression. Bound water did not correlate with any of the microarchitectural and compression parameters. We conclude that total water, T₁ and atomic space between the crystal and organic surface are altered in the trabecular bones of osteopenic rats, and PTH reverses these parameters. Furthermore, from these data, it appears that total water and T₁ could serve as trabecular surrogates of micro-architecture and compression strength.     

Linkages between phosphorus transformations and carbon decomposition in a forest soil

Phosphorus mineralization is chemically coupled with organic matter (OM) decomposition in surface horizons of a mixed-conifer forest soil from the Sierra Nevada, California, and is also affected by the disturbance caused by forest harvesting. Solution¹³C nuclear magnetic resonance (NMR) spectroscopy of NaOH extracts revealed a decrease of O-alkyl and alkyl-C fractions with increasing degree of decomposition and depth in the soil profile, while carbonyl and aromatic C increased. Solid-state¹³C-NMR analysis of whole soil samples showed similar trends, except that alkyl C increased with depth. Solution³¹P-NMR indicated that inorganic P (P₁) increased with increasing depth, while organic-P (P_o) fractions decreased. Close relationships between P mineralization and litter decomposition were suggested by correlations between P₁ and C fractions (r = 0.82, 0.81, –0.87, and –0.76 for carbonyl, aromatic, alkyl and O-alkyl fractions, respectively). Correlations for diester-P and pyrophosphate with O-alkyl (r = 0.63 and 0.84) and inverse correlations with aromatics (r = –0.74 and –0.72) suggest that mineralization of these P fractions coincides with availability of C substrate. A correlation between monoester P and alkyl C (r = 0.63) suggests mineralization is linked to breakdown of structural components of the plant litter. NMR analyses, combined with Hedley-P fractionation, suggest that post-harvest buildup of labile P in decomposed litter increases the potential for leaching of P during the first post-harvest season, but also indicates reduced biological activity that transports P from litter to the mineral soil. Thus, P is temporarily stored in decomposed litter, preventing its fixation by mineral oxides. In the mineral horizons,³¹P-NMR provides evidence of decline in biologically-available P during the first post-harvest season.     

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method.

The ultrastructural localization of alkaline phosphatase (A1P) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl2 to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl2 and CeCl3. A1P activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.     

Osteogenesis in cultures of limb mesenchymal cells

The results of previous reports demonstrated that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. The observations reported here suggest that initial cell plating densities may provide environmental conditions deterministic to a particular limb phenotype. Quantitative microscopic studies, histochemical localization of calcium phosphate, and electron microscopy indicate that osteoblasts develop in cultures derived from stage 24 limb mesenchymal cells. Additionally, 1–3% of the cells from stage 24 limbs are associated with mineral deposits when plated at initial high densities (5 × 10⁶ cells per 35-mm culture dish), while more than 50% of the cells are associated with cartilage by Day 9. Cultures plated at intermediate seeding densities (between 2.0 and 2.5 × 10⁶ cells per 35-mm culture dish) have minimal cartilage development, and approximately 20% of the cells are associated with mineral by Day 9. Furthermore, cultures prepared from stage 31 limb mesenchymal cells form well-developed bone nodules with both osteoblasts and osteocytes present, but no cartilage. It is clear from these observations and from a consideration of the initiation of osteogenesisin vivo that the initiation of bone development in the limb is not associated with cartilage development. Based on these studies and observations on the effect of nutrient factors on phenotypic expression in culture, an hypothesis is presented relating differential vascularization and nutrient flow to the determination of limb phenotypesin vivo.     

Model system studies of the influence of bacterial biofilm formation on mineral surface reactivity

Biofilm development on mineral surfaces and related changes in surface reactivity were studied using batch and flow through experiments. An artificial groundwater was used as the primary nutrient medium, Pseudomonas aeruginosa (PAO1) was the model microbial organism and ‘mineral’ surfaces were kept as simple as possible by using glass or a polished quartz tile. Experiments were also completed with very low concentrations (100 mg l^?1) of iron, Fe²⁺ , in the solution. In situ confocal laser scanning microscopy of developing colonies during the live growth phase, and of thick, mature biofilms, revealed only sporadic coverage of biofilm cells and associated polymers at the ‘mineral–microbe interface’. Imaging and analysis of biofilm-conditioned surfaces doped with Fe²⁺ -rich solutions allowed the locus and form of Fe-rich mineral precipitation to be determined and show that biological surface components can cause mineral precipitation from dilute dissolved species which might otherwise remain in solution.     

High-dimensionality 13C direct-detected NMR experiments for the automatic assignment of intrinsically disordered proteins

We present three novel exclusively heteronuclear 5D ¹³C direct-detected NMR experiments, namely (H^N-flipN)CONCACON, (HCA)CONCACON and (H)CACON(CA)CON, designed for easy sequence-specific resonance assignment of intrinsically disordered proteins (IDPs). The experiments proposed have been optimized to overcome the drawbacks which may dramatically complicate the characterization of IDPs by NMR, namely the small dispersion of chemical shifts and the fast exchange of the amide protons with the solvent. A fast and reliable automatic assignment of α-synuclein chemical shifts was obtained with the Tool for SMFT-based Assignment of Resonances (TSAR) program based on the information provided by these experiments.     

Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

In the presence of 4 mM inorganic phosphate, differentiating chick limb-bud mesenchymal cells plated in micromass cultures form a mineralized matrix resembling that of chick calcified cartilage. To test the hypothesis that cartilage proteoglycans are inhibitors of cell mediated mineralization, the synthesis, content, and turnover of proteoglycans were altered in this system, and the extent of mineralization and properties of the mineral crystals examined. In all cases where the proteoglycan synthesis or proteoglycans present were modified to provide fewer or smaller molecules, mineralization was enhanced. Specifically, when proteoglycan synthesis was blocked by treatment with 10⁻¹⁰ M retinoic acid, extensive mineral deposition occurred on a matrix devoid of both proteoglycans and cartilage nodules. The crystals, which formed rapidly, were relatively large in size based on analysis by X-ray diffraction or FT-IR microspectroscopy, and were more abundant than in controls. When 2.5 or 5 mM xylosides were used to cause the synthesis of smaller proteoglycans, the extent of mineral accretion was also increased relative to controls; however, the matrix was less affected, and the extent of mineral deposition and the size of the crystals were not as markedly altered as in the case of retinoic acid. Modification of existing proteoglycans by either chondroinase ABC or hyaluronidase treatment similarly resulted in increased mineral accretion (based on ⁴⁵Ca uptake or total Ca uptake) relative to cultures in which the proteoglycan content was not manipulated. Crystals were more abundant and larger than in control mineralizing cultures. In contrast, when proteoglycan degradation by metalloproteases was inhibited by metal chelation with o-phenanthroline, the Ca accretion at early time points was increased, but as mineralization progressed, Ca accumulation decreased. These data provide evidence that in this culture system, proteoglycans are inhibitors of mineralization. J. Cell. Biochem. 64:632–643. © 1997 Wiley-Liss, Inc.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.